CN106349389B - Tumor-specific anti-EGFR antibody and its application - Google Patents

Abstract

The present invention relates to tumor-specific anti-EGFR antibodies and applications thereof. The present invention discloses an antibody that specifically recognizes EGFRvIII in tumor cells or overexpressed EGFR but hardly recognizes EGFR in normal cells, which can be used to prepare various targeted antitumor drugs and drugs for tumor diagnosis.

Description

Tumor-specific anti-EGFR antibody and its application

technical field

The present invention belongs to the field of immunity, and more particularly, the present invention relates to tumor-specific anti-EGFR antibodies and applications thereof.

Background technique

EGFR is overexpressed or mutated in many tumors, and how to selectively identify these overexpressed or mutated EGFR is undoubtedly a very important scientific issue. So far, antibodies against the EGFR287-302 epitope are thought to recognize the overexpressed EGFR, EGFRvIII, and de4EGFR on the tumor surface, but do not recognize EGFR in normal cells. Unfortunately, in clinical trials of antibodies against this epitope, there will still be side effects such as skin rashes in patients (http://meetinglibrary.asco.org/content/115945-132), indicating that only targeting this epitope may still be effective. Recognizes EGFR in normal cells such as keratinocytes.

Therefore, how to screen higher tumor-specific anti-EGFR antibodies is very urgent. Antibodies with a high degree of tumor specificity have great potential application value whether they are used for tumor imaging diagnosis, individualized diagnosis or tumor targeted therapy.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide tumor-specific anti-EGFR antibodies and applications thereof.

In a first aspect of the present invention, there is provided an antibody that specifically recognizes EGFRvIII expressed by tumor cells or overexpressed EGFR, the antibody has a light chain variable region and a heavy chain variable region, and,

The CDR1 of its light chain variable region has an amino acid sequence selected from the group consisting of: SEQ ID NO:41, SEQ ID NO:47, SEQ ID NO:55;

The CDR2 of its light chain variable region has an amino acid sequence selected from the group consisting of: SEQ ID NO: 42, SEQ ID NO: 53;

The CDR3 of its light chain variable region has an amino acid sequence selected from the group consisting of: SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:57;

The CDR1 of its heavy chain variable region has the amino acid sequence: SEQ ID NO: 44;

The CDR2 of its heavy chain variable region has an amino acid sequence selected from the group consisting of: SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:52;

The CDR3 of its heavy chain variable region has an amino acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:50.

In a preferred embodiment, the antibody includes:

Antibody (a), whose light chain variable region has CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 42, and CDR3 shown in SEQ ID NO: 43; or its heavy chain variable region has CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 45, CDR3 shown in SEQ ID NO: 46;

Antibody (b), whose light chain variable region has CDR1 shown in SEQ ID NO: 47, CDR2 shown in SEQ ID NO: 42, and CDR3 shown in SEQ ID NO: 48; or its heavy chain variable region has CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 49, CDR3 shown in SEQ ID NO: 50;

Antibody (c), whose light chain variable region has CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 42, and CDR3 shown in SEQ ID NO: 48; or its heavy chain variable region has CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 51, CDR3 shown in SEQ ID NO: 50;

Antibody (d), whose light chain variable region has CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 42, and CDR3 shown in SEQ ID NO: 43; or its heavy chain variable region has CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 52, CDR3 shown in SEQ ID NO: 50;

Antibody (e), whose light chain variable region has CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 42, and CDR3 shown in SEQ ID NO: 43; or its heavy chain variable region has CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 45, CDR3 shown in SEQ ID NO: 50;

Antibody (f), whose light chain variable region has CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 53, and CDR3 shown in SEQ ID NO: 54; or its heavy chain variable region has CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 51, CDR3 shown in SEQ ID NO: 50;

Antibody (g), whose light chain variable region has CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 42, and CDR3 shown in SEQ ID NO: 54; or its heavy chain variable region has CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 51, CDR3 shown in SEQ ID NO: 50;

Antibody (h), whose light chain variable region has CDR1 shown in SEQ ID NO: 55, CDR2 shown in SEQ ID NO: 42, and CDR3 shown in SEQ ID NO: 56; or its heavy chain variable region has CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 45, CDR3 shown in SEQ ID NO: 50;

Antibody (i), whose light chain variable region has CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 53, and CDR3 shown in SEQ ID NO: 56; or its heavy chain variable region has CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 52, CDR3 shown in SEQ ID NO: 50;

Antibody (j), whose light chain variable region has CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 42, and CDR3 shown in SEQ ID NO: 56; or its heavy chain variable region has CDR1 shown in SEQ ID NO: 44, CDR2 shown in SEQ ID NO: 52, CDR3 shown in SEQ ID NO: 50;

Antibody (k), whose light chain variable region has CDR1 shown in SEQ ID NO: 41, CDR2 shown in SEQ ID NO: 42, and CDR3 shown in SEQ ID NO: 57; or its heavy chain variable region has CDR1 set forth in SEQ ID NO:44, CDR2 set forth in SEQ ID NO:52, CDR3 set forth in SEQ ID NO:50; or

The antibody (1) recognizes the same epitope as the epitope recognized by the antibody according to any one of (a) to (k).

In another preferred example, the specific recognition of EGFRvIII expressed by tumor cells or the overexpressed EGFR can be: single chain antibody (scFV), monoclonal antibody, domain antibody, Fab fragment, Fd fragment, Fv fragment, F(ab') ₂ fragments and derivatives thereof, or other forms of antibodies; preferably single chain antibodies.

In another preferred embodiment, the antibody that specifically recognizes EGFRvIII expressed by tumor cells or overexpressed EGFR is humanized, fully human, chimeric or murine.

In another preferred embodiment, the amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 of SEQ ID NO: 13; or the amino acid sequence of the variable region of the light chain of the antibody is as follows Shown at positions 1-108 in SEQ ID NO: 13;

The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 59; or the amino acid sequence of the variable region of the light chain of the antibody is shown in position 124-239 in SEQ ID NO: 59. 1-108 bits are shown;

The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 61; or the amino acid sequence of the variable region of the light chain of the antibody is shown in position 124-239 of SEQ ID NO: 61. 1-108 bits are shown;

The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 63; or the amino acid sequence of the variable region of the light chain of the antibody is shown in position 124-239 of SEQ ID NO: 63. 1-108 bits are shown;

The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 65; or the amino acid sequence of the variable region of the light chain of the antibody is shown in position 124-239 of SEQ ID NO: 65. 1-108 bits are shown;

The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 67; or the amino acid sequence of the variable region of the light chain of the antibody is shown in position 124-239 of SEQ ID NO: 67. 1-108 bits are shown;

The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 69; or the amino acid sequence of the variable region of the light chain of the antibody is shown in position 124-239 of SEQ ID NO: 69. 1-108 bits are shown;

The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 71; or the amino acid sequence of the variable region of the light chain of the antibody is shown in position 124-239 of SEQ ID NO: 71. 1-108 bits are shown;

The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 73; or the amino acid sequence of the variable region of the light chain of the antibody is shown in position 124-239 of SEQ ID NO: 73. 1-108 bits are shown;

The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 75; or the amino acid sequence of the variable region of the light chain of the antibody is shown in position 124-239 of SEQ ID NO: 75. 1-108 bits indicated; or

The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 77; or the amino acid sequence of the variable region of the light chain of the antibody is shown in position 124-239 of SEQ ID NO: 77. 1-108 bits are shown.

In another preferred embodiment, the antibody is antibody (a); more preferably, the amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 13; or The amino acid sequence of the light chain variable region of the antibody is shown in positions 1-108 of SEQ ID NO: 13.

In another aspect of the invention, nucleic acids encoding the aforementioned antibodies are provided.

In another aspect of the present invention, an expression vector is provided, which comprises the nucleic acid. In another preferred embodiment, the expression vector is a PH/DHFR vector.

In another aspect of the present invention, a host cell is provided, which comprises the expression vector or the nucleic acid integrated into the genome. In another preferred embodiment, the host cells are eukaryotic host cells or prokaryotic host cells; preferably eukaryotic host cells, more preferably Chinese hamster ovary cells (CHO).

In another aspect of the present invention, there is provided the use of any one of the aforementioned antibodies, for the preparation of targeted drugs, antibody drug conjugates or multifunctional antibodies that specifically target tumor cells expressing EGFRvIII or overexpressed EGFR Antibodies; or reagents for the preparation of diagnosing tumors, the tumors express EGFRvIII or overexpressed EGFR; or for the preparation of chimeric antigen receptor-modified immune cells; preferably, the immune cells include: T lymphocytes, NK cells cells or NKT lymphocytes.

In another aspect of the present invention, there is provided a multifunctional immunoconjugate, the multifunctional immunoconjugate comprises: any one of the aforementioned antibodies; and linked thereto (including covalently linked, coupled, Adhesion, adsorption) functional molecules; the functional molecules are selected from: molecules targeting tumor surface markers, tumor-inhibiting molecules, molecules targeting immune cell surface markers or detectable markers.

In a preferred embodiment, the molecules targeting tumor surface markers are antibodies or ligands that bind to tumor surface markers; or the tumor-inhibiting molecules are anti-tumor cytokines or anti-tumor toxins; Preferably, the cytokines include (but are not limited to): IL-12, IL-15, IFN-beta, TNF-alpha.

In another preferred example, in the multifunctional immunoconjugate, the detectable label includes: a fluorescent label and a chromogenic label.

In another preferred example, in the multifunctional immunoconjugate, the molecule targeting the surface marker of immune cells is an antibody or ligand that binds to the surface marker of immune cells; preferably, the The immune cell surface markers include (but are not limited to): CD3, CD16, CD28.

In another preferred example, in the multifunctional immunoconjugate, the molecule targeting the surface marker of immune cells is an antibody that binds to the surface marker of T cells, which is similar to any of the aforementioned antibodies. Bispecific T cell engager (BiTE) is formed.

In another preferred embodiment, in the multifunctional immunoconjugate, the antibody that binds to an immune cell surface marker is an anti-CD3 antibody. In another preferred embodiment, the anti-CD3 antibody is a single chain antibody (scFV), monoclonal antibody, Fab fragment, Fd fragment, Fv fragment, F(ab') ₂ fragment and its derivatives, or other forms of Antibodies; preferably single chain antibodies. In another preferred embodiment, the anti-CD3 antibody is humanized, fully human, chimeric or murine.

In another preferred embodiment, the multifunctional immunoconjugate is a fusion polypeptide, and a linking peptide (linker) is also included between any of the aforementioned antibodies and the functional molecule linked thereto.

In another preferred embodiment, the sequence of the connecting peptide is (GlyGlyGlyGlySer)n, wherein n is an integer from 1 to 5; more preferably, n=3.

In another preferred embodiment, the multifunctional immunoconjugate is administered in the form of polypeptide or by gene administration.

In another aspect of the present invention, nucleic acids encoding the multifunctional immunoconjugates are provided.

In another aspect of the present invention, there is provided the use of any of the multifunctional immunoconjugates described above, for the preparation of antitumor drugs, or for the preparation of reagents for diagnosing tumors, the tumors expressing EGFRvIII or overexpressed EGFR; Or used to prepare immune cells modified with chimeric antigen receptors; preferably, the immune cells include: T lymphocytes, NK cells or NKT lymphocytes.

In another aspect of the present invention, there is provided a chimeric antigen receptor comprising any of the aforementioned antibodies, which is expressed on the surface of an immune cell, the chimeric antigen receptor comprising sequentially linked: any of the aforementioned antibodies Antibody, transmembrane region and intracellular signal region; the intracellular signal region is selected from: CD3ζ, FceRIγ, CD27, CD28, CD137, CD134 intracellular signal region sequence, or a combination thereof.

In a preferred embodiment, the transmembrane region comprises the transmembrane region of CD8 or CD28.

In another preferred embodiment, the immune cells include: T lymphocytes, NK cells or NKT cells.

In another preferred embodiment, the chimeric antigen receptor comprises the following sequence-linked antibodies, a transmembrane region and an intracellular signal region:

any of the foregoing antibodies, CD8 and CD3ζ;

any of the foregoing antibodies, CD8, CD137 and CD3ζ;

any of the foregoing antibodies, the transmembrane region of the CD28 molecule, the intracellular signaling region of the CD28 molecule, and CD3ζ; or

Any of the aforementioned antibodies, the transmembrane region of the CD28 molecule, the intracellular signaling region of the CD28 molecule, CD137 and CD3ζ.

In another preferred embodiment, the antibody is a single-chain antibody or a domain antibody.

In another preferred embodiment, the chimeric antigen receptor has:

SEQ ID NO: 36 or the amino acid sequence shown at positions 285-601 therein; or

SEQ ID NO: 37 or the amino acid sequence shown at positions 285-702 therein; or

SEQ ID NO: 38 or the amino acid sequence shown at positions 285-744 therein; or

SEQ ID NO: 39 or the amino acid sequence shown at positions 285-749 therein; or

SEQ ID NO: 40 or the amino acid sequence shown at positions 285-791 therein.

In another aspect of the invention, there is provided a nucleic acid encoding any of the foregoing chimeric antigen receptors. In another preferred embodiment, the nucleic acid encoding the chimeric antigen receptor has:

SEQ ID NO: 31 or the nucleotide sequence of paragraphs 966-1916 therein; or

SEQ ID NO: 32 or the nucleotide sequence set forth in paragraphs 966-2219 therein; or

SEQ ID NO: 33 or the nucleotide sequence set forth in paragraphs 966-2345 therein; or

SEQ ID NO: 34 or the nucleotide sequence of paragraphs 966-2360 therein; or

SEQ ID NO: 35 or the nucleotide sequence set forth at 966-2486 therein.

In another aspect of the present invention, there is provided an expression vector comprising the aforementioned nucleic acid.

In another preferred embodiment, the expression vector is derived from the lentiviral plasmid pWPT (or pWPT-eGFP).

In another aspect of the present invention, there is provided a virus comprising the aforementioned vector.

Use of any of the aforementioned chimeric antigen receptors or nucleic acid encoding the same, or an expression vector or virus containing the nucleic acid, for preparing genetically modified immune cells targeting tumor cells expressing EGFRvIII or overexpressing EGFR.

In another aspect of the present invention, a genetically modified immune cell is provided, which is transduced with the nucleic acid, or the expression vector or the virus; or the surface of which expresses the chimeric antigen receptor .

In a preferred example, the immune cells also carry the coding sequences of exogenous cytokines; preferably, the cytokines include: IL-12, IL-15 or IL-21.

In another preferred example, the immune cell also expresses another chimeric antigen receptor, the receptor does not contain CD3ζ, but contains the intracellular signaling domain of CD28, the intracellular signaling domain of CD137, or the like. a combination of the two.

In another preferred embodiment, the immune cells also express chemokine receptors; preferably, the chemokine receptors include: CCR2.

In another preferred embodiment, the immune cells also express siRNA that can reduce the expression of PD-1 or a protein that blocks PD-L1.

In another preferred example, the immune cells also express a safety switch; preferably, the safety switch includes: iCaspase-9, Truancated EGFR or RQR8.

In another aspect of the present invention, the use of the genetically modified immune cells is provided for preparing a drug for inhibiting tumors, where the tumor is a tumor expressing EGFRvIII or overexpressing EGFR.

In another aspect of the present invention, there is provided a pharmaceutical composition (including a medicament or a diagnostic reagent) comprising:

any of the foregoing antibodies or a nucleic acid encoding the antibody; or

Any of the aforementioned immunoconjugates or a nucleic acid encoding the conjugate; or

Any of the aforementioned chimeric antigen receptors or a nucleic acid encoding the chimeric antigen receptor; or

The genetically modified immune cell of any of the foregoing.

Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein.

Description of drawings

Figure 1. Antibodies 7B3 and Y022 can specifically bind to antigens EGFRvIII and N1N2-806 (phage ELISA experiment).

Figure 2. Binding curve of antibody 7B3 to antigen EGFRvIII.

Figure 3. Binding curve of antibody Y022 to antigen EGFRvIII.

Figure 4. Electropherograms of purification of three scFv-Fc fusion antibodies.

Figure 5. FACS detection of the binding ability of single chain antibodies scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc to cell surface EGFR.

Figure 6. Schematic diagram of the structure of pH-Y022/CD3 expression vector.

Figure 7. SDS-PAGE detection of single chain diabodies Y022/CD3, 806/CD3 and C225/CD3.

Figure 8. The antigen-binding specificity of Y022/CD3 single-chain bifunctional antibody was detected by FACS.

Figure 9. Cytotoxicity profile of single chain diabodies.

Figure 10. Schematic diagram of the connection sequence of each part of the chimeric antigen receptor.

Detailed ways

After in-depth research and screening, the present inventors obtained an antibody that specifically recognizes EGFRvIII in tumor cells or overexpressed EGFR, but hardly recognizes EGFR in normal cells. The antibody of the present invention can be applied to prepare various targeted antitumor drugs and drugs for tumor diagnosis.

anti-EGFR antibody

The inventors further screened and mutated amino acids based on the humanized antibodies obtained earlier, and found an anti-EGFR antibody that can more specifically target EGFR in tumor cells, which selectively binds to tumors that overexpress EGFR or EGFRvIII , while not binding to normal cell EGFR.

The antibody of the present invention can be a complete immunoglobulin molecule, or can be an antigen-binding fragment, including but not limited to Fab fragment, Fd fragment, Fv fragment, F(ab') ₂ fragment, complementarity determining region (CDR) fragment, single Chain antibody (scFv), domain antibody, bivalent single chain antibody, single chain phage antibody, bispecific diabody, trichain antibody, tetrabody.

The antigen-binding properties of antibodies can be described by three specific regions located in the variable regions of the heavy and light chains, called complementarity determining regions (CDRs), which separate the variable regions into four Framework region (FR), the amino acid sequence of the four FRs is relatively conservative and does not directly participate in the binding reaction. These CDRs form a circular structure, and the β-sheets formed by the FRs in between are spatially close to each other, and the CDRs on the heavy chain and the CDRs on the corresponding light chain constitute the antigen-binding site of the antibody. Which amino acids make up the FR or CDR regions can be determined by comparing the amino acid sequences of antibodies of the same type. The CDR regions are sequences of proteins of immunological interest, and the CDR regions of the antibodies of the present invention are novel. The antibody may comprise two, three, four, five or all six CDR regions disclosed herein.

Another aspect of the invention includes functional variants of the antibodies described herein. If the variant can compete with the parent antibody for specific binding to SEQ ID NO: 1, and its ability to recognize tumor cell EGFRvIII or overexpressed EGFR is close to the specific antibody provided in the examples of the present invention. The functional variants may have conservative sequence modifications, including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as directed mutagenesis and random PCR-mediated mutagenesis, and can include natural as well as non-natural nucleotides and amino acids. Preferably, the modification of the sequence occurs in regions other than the CDR regions of the antibody.

immunoconjugate

The invention also provides multifunctional immunoconjugates comprising the antibodies described herein and further comprising at least one other type of functional molecule. The functional molecules are selected from, but not limited to, molecules targeting tumor surface markers, molecules that inhibit tumors, molecules targeting surface markers of immune cells, or detectable markers. The antibody and the functional molecule can form a conjugate by covalent connection, coupling, attachment, cross-linking and the like.

As a preferred mode, the immunoconjugate may comprise: the antibody of the present invention and at least one molecule targeting tumor surface markers or tumor-inhibiting molecules. The tumor-inhibiting molecules can be anti-tumor cytokines, or anti-tumor toxins; preferably, the cytokines include (but are not limited to): IL-12, IL-15, IFN-beta, TNF -alpha. The molecules targeting tumor surface markers, for example, can synergize with the antibodies of the present invention to more precisely target tumor cells.

As a preferred mode, the immunoconjugate may comprise: the antibody of the present invention and a detectable label. The detectable labels include but are not limited to: fluorescent labels, chromogenic labels; such as: enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron-emitting metals and non-radioactive paramagnetic Metal ion. More than one marker may also be included. The labels used to label antibodies for detection and/or analysis and/or diagnostic purposes depend on the particular detection/analysis/diagnostic technique and/or method used eg immunohistochemical staining of (tissue) samples, flow cytometry, etc. Suitable labels for detection/analysis/diagnostic techniques and/or methods known in the art are well known to those skilled in the art.

As a preferred mode, the immunoconjugate may comprise: the antibody of the present invention and a molecule targeting a surface marker of an immune cell. The molecule targeting the surface marker of immune cells can recognize immune cells, which carry the antibody of the present invention to reach immune cells, and at the same time, the antibody of the present invention can target immune cells to tumor cells, thereby causing immune cells to specifically kill tumor.

As a means of chemically producing immunoconjugates by direct or indirect conjugation (eg, through a linker), the immunoconjugates can be produced as fusion proteins comprising the antibodies of the invention and suitable other protein. Fusion proteins can be produced by methods known in the art, such as recombinantly by constructing a nucleic acid molecule comprising an in-frame nucleotide sequence encoding an antibody and a nucleoside encoding a suitable label and subsequent expression of the nucleic acid molecule acid sequence.

Another aspect of the invention provides nucleic acid molecules encoding at least one antibody, functional variant or immunoconjugate thereof of the invention. Once the relevant sequences have been obtained, recombinant methods can be used to obtain the relevant sequences in bulk. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods.

The present invention also relates to vectors comprising suitable DNA sequences as described above together with suitable promoter or control sequences. These vectors can be used to transform appropriate host cells so that they can express proteins. Host cells can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells.

Chimeric Antigen Receptors and Genetically Modified Immune Cells

The present invention provides a chimeric antigen receptor expressed on the surface of immune effector cells (immune cells). wherein the extracellular binding region comprises an antibody of the present invention. Expressing the chimeric antigen receptor on the surface of immune effector cells can make the immune effector cells have a highly specific cytotoxic effect on tumor cells expressing EGFRvIII or overexpressing EGFR.

As used herein, "immune cells" and "immune effector cells" are used interchangeably and include: T lymphocytes, NK cells or NKT cells, and the like.

As a preferred mode of the present invention, the antibody contained in the chimeric antigen receptor is a single-chain antibody, which is connected to the transmembrane region of CD8 or CD28 through the CD8 hinge region, and the transmembrane region is followed by an intracellular signal. Area.

The present invention also includes nucleic acids encoding such chimeric antigen receptors. The present invention also relates to variants of the above-mentioned polynucleotides, which encode polypeptides or fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention.

The transmembrane region of the chimeric antigen receptor can be selected from the transmembrane regions of proteins such as CD8 or CD28. Human CD8 protein is a heterodimer composed of αβ or γδ chains. In one embodiment of the invention, the transmembrane region is selected from the transmembrane region of CD8α or CD28. In addition, the CD8α hinge region (hinge) is a flexible region, therefore, CD8 or CD28 and the transmembrane region plus the hinge region are used to connect the target recognition domain scFv and intracellular signaling region of the chimeric antigen receptor CAR .

The intracellular signaling region may be selected from the group consisting of CD3ζ, FcεRIγ, CD28, CD137, the intracellular signaling region of CD134 proteins, and combinations thereof. The CD3 molecule consists of five subunits, of which the CD3ζ subunit (also known as CD3zeta, Z for short) contains three ITAM motifs, which are important signal transduction regions in the TCR-CD3 complex. CD3δZ is a truncated CD3ζ sequence that does not have an ITAM motif and is generally used for the construction of a negative control in the practice of the present invention. FcεRIγ is mainly distributed on the surface of mast cells and basophils, and it contains an ITAM motif, which is similar to CD3ζ in structure, distribution and function. In addition, as mentioned above, CD28, CD137, and CD134 are costimulatory signal molecules. After binding to their respective ligands, the costimulatory effect produced by their intracellular signal segments causes the continuous proliferation of immune effector cells (mainly T lymphocytes). And it can increase the level of cytokines such as IL-2 and IFN-γ secreted by immune effector cells, and at the same time improve the survival cycle and anti-tumor effect of CAR immune effector cells in vivo.

The chimeric antigen receptors of the present invention can be linked sequentially as follows:

Antibodies of the invention, CD8 and CD3ζ;

Antibodies of the invention, CD8, CD137 and CD3ζ;

An antibody of the invention, a transmembrane region of a CD28 molecule, an intracellular signaling region of a CD28 molecule, and CD3ζ; or

Antibody of the present invention, transmembrane region of CD28 molecule, intracellular signal region of CD28 molecule, CD137 and CD3ζ.

and combinations thereof, wherein CD28a in the related chimeric antigen receptor protein represents the transmembrane region of the CD28 molecule, and CD28b represents the intracellular signal region of the CD28 molecule. The various chimeric antigen receptors mentioned above are collectively referred to as scFv(EGFR)-CAR.

The present invention also provides a vector comprising the above-mentioned nucleic acid encoding the chimeric antigen receptor protein expressed on the surface of immune effector cells. In a specific embodiment, the vector used in the present invention is a lentiviral plasmid vector pWPT-eGFP. This plasmid belongs to the third-generation self-inactivating lentiviral vector system. The system has three plasmids, namely, the encoding protein Gag/Pol, the packaging plasmid psPAX2 encoding the Rev protein; the envelope plasmid PMD2.G encoding the VSV-G protein; and the empty The vector pWPT-eGFP can be used for recombinant introduction of the nucleic acid sequence of interest, that is, the nucleic acid sequence encoding the CAR. The expression of enhanced green fluorescent protein (eGFP) is regulated by the elongation factor-1α (EF-1α) promoter in the empty vector pWPT-eGFP. The recombinant expression vector pWPT-eGFP-F2A-CAR containing the target nucleic acid sequence encoding CAR is produced by the ribosomal skipping sequence 2A (F2A for short) from the foot-and-mouth disease virus (FMDV). ) to achieve co-expression of eGFP and CAR.

The present invention also includes viruses comprising the above-described vectors. The virus of the present invention includes the packaged infectious virus, and also includes the virus to be packaged containing the necessary components for packaging as the infectious virus. Other viruses and their corresponding plasmid vectors known in the art that can be used for the transduction of exogenous genes into immune effector cells can also be used in the present invention.

The present invention also provides genetically modified immune effector cells, which are transduced with the nucleic acid of the present invention or the above-mentioned recombinant plasmid of the present invention comprising the nucleic acid, or a virus comprising the plasmid. Conventional nucleic acid transduction methods in the art, including non-viral and viral transduction methods, can be used in the present invention. Non-viral-based transduction methods include electroporation and transposon methods. Recently, the Nucleofector nucleotransfection instrument developed by Amaxa can directly introduce foreign genes into the nucleus to obtain efficient transduction of target genes. In addition, the transduction efficiency based on the Sleeping Beauty transposon (Sleeping Beautysystem) or the PiggyBac transposon and other transposon systems has been greatly improved compared with ordinary electroporation. It has been reported [Davies JK., et al. Combining CD19 redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies. Cancer Res, 2010, 70(10): OF1-10.], this method not only It has high transduction efficiency and can achieve site-directed integration of target genes. In one embodiment of the present invention, the method of transduction to effect chimeric antigen receptor gene-modified immune effector cells is a virus-based transduction method such as retrovirus or lentivirus. The method has the advantages of high transduction efficiency, stable expression of exogenous genes, and shortening the time for in vitro cultured immune effector cells to reach clinical level. On the surface of the transgenic immune effector cell, the transduced nucleic acid is expressed on its surface through transcription and translation. It is proved by in vitro cytotoxicity experiments on various cultured tumor cells that the immune effector cells of the present invention have a highly specific tumor cell killing effect (also known as cytotoxicity). Therefore, the nucleic acid encoding the chimeric antigen receptor protein, the plasmid containing the nucleic acid, the virus containing the plasmid and the transgenic immune effector cells transduced with the nucleic acid, plasmid or virus of the present invention can be effectively used for tumor immunotherapy.

The immune cells of the present invention can also carry the coding sequences of exogenous cytokines; the cytokines include but are not limited to: IL-12, IL-15 or IL-21 and the like. These cytokines have immunomodulatory or anti-tumor activities, can enhance the function of effector T cells and activated NK cells, or directly exert anti-tumor effects. Therefore, those skilled in the art can understand that the use of these cytokines helps the immune cells to function better.

The immune cells of the present invention can also express another chimeric antigen receptor other than the above-mentioned chimeric antigen receptor, the receptor does not contain CD3ζ, but contains the intracellular signal domain of CD28 and the intracellular signal of CD137 domain or a combination of the two.

The immune cells of the present invention can also express chemokine receptors; the chemokine receptors include but are not limited to CCR2. Those skilled in the art can understand that the CCR2 chemokine receptor can make CCR2 in vivo competitively bind with it, which is beneficial for blocking tumor metastasis.

The immune cells of the present invention can also express siRNA that can reduce the expression of PD-1 or a protein that blocks PD-L1. Those skilled in the art can understand that competitive blocking of the interaction of PD-L1 with its receptor PD-1 is beneficial to restore anti-tumor T cell responses, thereby inhibiting tumor growth.

The immune cells of the present invention can also express a safety switch; preferably, the safety switch includes: iCaspase-9, Truancated EGFR or RQR8.

pharmaceutical composition

The antibodies, immunoconjugates comprising the antibodies, and genetically modified immune cells of the present invention can be applied to the preparation of pharmaceutical compositions or diagnostic reagents. In addition to comprising an effective amount of the antibody, immunoconjugate or immune cell, the composition may also comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.

Specific examples of some substances which may be pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as carboxymethyl cellulose Sodium, ethyl cellulose and methyl cellulose; tragacanth powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, Sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerol, sorbitol, mannitol, and polyethylene glycols; alginic acid; emulsifiers such as Wetting agents, such as sodium lauryl sulfate; coloring agents; flavoring agents; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffers, among others.

The composition of the present invention can be prepared into various dosage forms according to needs, and can be administered by a physician at a dose beneficial to the patient according to factors such as the type, age, weight and general disease state of the patient, and the mode of administration. The mode of administration can be, for example, by injection or other treatment.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental methods that do not indicate specific conditions in the following examples are usually in accordance with conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, 2002, or according to the conditions described by the manufacturer. the proposed conditions.

Example 1. Construction of single-chain antibody 7B3 affinity maturation library

Single-chain antibody 7B3 is a humanized antibody fragment that specifically recognizes the cryptic epitope formed by the amino acid sequence 287-302 of EGFR exposed in tumor cells ( ²⁸⁷ CGADSYEMEEDGVRKC ³⁰² (SEQ ID NO: 1) ). The nucleotide sequences of its VL and VH genes are obtained from the sequences SEQ ID NO: 14 and SEQ ID NO: 13 shown in the patent 201210094008.x, and are connected in the sequence of VL _7B3 -linker-VH _7B3 .

Nucleotide sequence of single chain antibody 7B3 (717 base pairs, SEQ ID NO: 2):

The amino acid sequence of single-chain antibody 7B3 (239 amino acids, SEQ ID NO: 3; the underlined regions are 7B3VLCDR1, CDR2, CDR3, 7B3VH CDR1, CDR2, CDR3 in sequence):

In order to improve the binding ability of 7B3 single-chain antibody to EGFR, random mutations were performed on part of amino acids in the light chain CDR3 and heavy chain CDR3 regions, respectively, and the corresponding affinity maturation library was constructed.

1. Construction of 7B3 light chain CDR3 affinity maturation library

Through sequence alignment and analysis of the 7B3 single-chain antibody, some amino acids in the third CDR region of the 7B3 light chain were selected, and random mutations were introduced through primers to construct a light chain affinity maturation library.

In order to prepare the DNA fragments encoding the 7B3 mutant library, using the plasmid pCantab 5E-7B3 (insert 7B3 into the sfiI/NotI site of pCantab 5E-7B3) as a template, two DNA fragments were obtained by PCR method, followed by bridging. spliced by PCR. Specifically, the following steps were used: To synthesize the gene, carry out a PCR reaction in a volume of 50 μl, each reaction used plasmid pCantab 5E-7B3 as a template, the final concentration of each primer was 0.2 μM, and 5 μl 10×KOD Plus buffer, 4 μl dNTPs (dATP, dCTP, dGTP and dTTP, 2 mM each), 2 μl of 25 mM MgSO4 and 1 UKOD Plus (from Takara), after making up to volume with water, started the PCR program in a thermal cycler. The reaction was pre-denatured by heating the samples to 94°C for 5 minutes, followed by incubation for 25 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 68°C for 30 seconds. The final incubation was at 68°C for 10 minutes. The first fragment was amplified using primers pC7B3fw (SEQ ID NO: 4, ATAACAGGCCCAGCCGGCCATGGATATTCAGATGACCCAGAG) and LR3re (SEQ ID NO: 5, CACTTTGGTGCCCTGGCCAAATGTMNNTGGGNNMN NMNNMNNCTGMNNGCAATAATAGGTCGCAAAATC) and the second fragment using primers LR3f2fw (SEQ ID NO: 6, ACATTTGGCCAGGGCACCAAAG) and pC7B3re SEQ ID NO: 7, ATAAATGCGGCCGCGCTGCTCACGGTCAC).

The expected PCR products were identified by analytical agarose gel electrophoresis and recovered from samples purified using Wizard SV Gel and PCR Clean-up kits (purchased from Promega). The two fragments were added in an equimolar ratio to the second round of bridging PCR as a template. The reaction system still used the KOD Plus system mentioned above. The reaction was first heated to 94 °C for pre-denaturation for 5 minutes, and then incubated for 10 cycles. Cyclic reaction conditions were 94°C for 30 seconds, 60°C for 30 seconds, and 68°C for 30 seconds. The final incubation was at 68°C for 10 minutes. Subsequently, primers pC7B3fw and pC7B3re at a final concentration of 0.2 μM were directly added to the reaction system, and the PCR program was started. The reaction was pre-denatured by heating the samples to 94°C for 5 minutes, followed by incubation for 25 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 68°C for 30 seconds. The final incubation was at 68°C for 10 minutes. The expected PCR products were separated by preparative agarose gel electrophoresis and purified and recovered using the Wizard SV Gel and PCR Clean-up kit according to the manufacturer's instructions.

The complete library DNA fragment contains sfiI and NotI restriction endonuclease recognition sites at both ends, respectively, which were digested with restriction endonucleases sfiI/NotI (purchased from New England Biolabs), and inserted into the same double-enzyme cut. Phagemid vector pCANTAB 5E. The ligation product was isolated from the DNA in the samples using the Wizard SV Gel and PCR Clean-up kit and desalted for electroporation. During electrotransformation, an electroporation cup and electroporator Gene Pulser II (purchased from Bio-Rad) were used to transform into home-made competent E. coli ER2738 (purchased from New England Biolabs). The final confirmation resulted in a library containing ^1.9x109 mutants.

2. Construction of 7B3 heavy chain CDR3 affinity maturation library

Through sequence alignment and analysis of the 7B3 single-chain antibody, some amino acids in the third CDR region of the 7B3 heavy chain were selected, and random mutations were introduced through primers to construct a heavy chain affinity mature mutant library.

In order to prepare the DNA fragments encoding the 7B3 mutant library, using plasmid pCantab 5E-7B3 as the template, two DNA fragments were obtained by PCR method, and then spliced by bridging PCR method. Specifically, the following steps were used: To synthesize the gene, a PCR reaction was performed in a volume of 50 μl, each reaction used the plasmid pCantab 5E-7B3 as a template, the final concentration of each primer was 0.2 μM, and 5 μl 10×KOD Plus buffer, 4 μl dNTPs (dATP, dCTP, dGTP and dTTP, 2 mM each), 2 μl of 25 mM MgSO ₄ and 1 U KOD Plus, after making up the volume with water, the PCR program was started in a thermal cycler. The reaction was pre-denatured by heating the samples to 94°C for 5 minutes, followed by incubation for 25 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 68°C for 30 seconds. The final incubation was at 68°C for 10 minutes. The first fragment was amplified using primers HR3f1fw (SEQ ID NO: 8, TCGCAATTCCTTTAGTTGTTCC) and HR3f1re (SEQ ID NO: 9, CAGGGTGCCCTGGCCCCAGTAANNMNNMNNMNNMNNMNNGCGCGCGCAATAATACAC) and the second fragment using primers HR3f2fw (SEQ ID NO: 10, TACTGGGGCCAGGGCACCCTG) and HR3f2re ( SEQ ID NO: 11, GGAATAGGTGTATCACCGTACTCAG).

The expected PCR product was identified by analytical agarose gel electrophoresis and recovered from the samples using the Wizard SV Gel and PCR Clean-up kit. The two fragments were added in an equimolar ratio to the second round of bridging PCR as a template. The reaction system still used the KOD Plus system mentioned above. In the absence of primers, the reaction was first heated to 94 ℃ for 5 minutes. Pre-denaturation, This was followed by 10 cycles of incubation, each cycle of reaction conditions being 94°C for 30 seconds, 60°C for 30 seconds and 68°C for 30 seconds. The final incubation was at 68°C for 10 minutes. Subsequently, primers HR3f1fw and HR3f2re at a final concentration of 0.2 μM were directly added to the reaction system, and the PCR procedure was started. The reaction was pre-denatured by heating the samples to 94°C for 5 minutes, followed by incubation for 25 cycles of 94°C for 30 seconds, 56°C for 30 seconds, and 68°C for 30 seconds. The final incubation was at 68°C for 10 minutes. The expected PCR products were separated by preparative agarose gel electrophoresis and purified and recovered using the Wizard SV Gel and PCR Clean-up kit according to the manufacturer's instructions.

The complete library DNA fragment contains sfiI and NotI restriction endonuclease recognition sites at both ends, which are digested with restriction endonucleases sfiI/NotI, and inserted into the phagemid vector pCANTAB5E that has undergone the same double digestion. The ligation product was isolated from the DNA in the samples using the Wizard SV Gel and PCR Clean-up kit and desalted for electroporation. During electrotransformation, the electroporation cup and electroporator Gene Pulser II were used to transform into home-made competent Escherichia coli ER2738. It was finally confirmed that a library containing 6.0 x ¹⁰⁹ mutants was obtained.

Example 2. Screening against EGFRvIII using 7B3 affinity maturation library

In order to obtain the 7B3 mutant with higher affinity, the light chain and heavy chain mutant libraries were used for four rounds of screening, respectively. The phage library was incubated with the biotin-labeled antigen EGFRvIII (purchased from Ruijin Biotech) at room temperature for 2 hours, and then mixed with 2% (w/v) BSA (bovine serum albumin, purchased from Shanghai Sangong) in the blocking solution. The blocked streptavidin magnetic beads MyOne C1 (purchased from Invitrogen) were incubated at room temperature for 30 minutes. The magnetic beads were then washed with PBST (containing 0.1% Tween-20) buffer to remove non-specific binding or weakly binding phage. The phage with strong binding ability was eluted from the magnetic beads with glycine-hydrochloric acid (pH 2.2), neutralized with Tris neutralization solution (pH 9.1), and used to infect E. coli ER2738 in the mid-logarithmic growth phase, and was used for the next round of screening.

In the above four rounds of screening, the amount of magnetic beads was 50 μl, 25 μl, 10 μl and 10 μl, the concentration of biotin-labeled antigen EGFRvIII was 10 nM, 1 nM, 0.5 nM and 0.1 nM, respectively, and the washing times of PBST were 10 times and 10 times, respectively. times, 15 times and 20 times. Starting from the second round of screening, 50-fold, 500-fold and 1000-fold excess of the non-biotinylated antigen EGFRvIII was added for competition before elution to remove mutants with weaker binding ability.

For the production of phages displaying the 7B3 single chain antibody mutant on the surface, the glycerol bacteria obtained in Example 1 were inoculated in 400 ml of 2YT/ampicillin medium to bring the cell density to OD ₆₀₀ = 0.1, at 37° C. and 200 rpm The culture was shaken until the cell density reached _OD600 = 0.5. The cells were infected with 10 ¹² pfu of M13KO7 helper phage and incubated at 30°C and 50 rpm for 30 minutes. After adding 50 mg/1 kanamycin, incubate with shaking at 37°C and 200rpm for 30 minutes, separate the pellet by centrifugation (15 minutes, 1600×g, 4°C), and resuspend in 400ml of 2YT/ampicillin/kanamycin Supplementary medium was cultured at 37°C and 200 rpm for 16 hours with shaking. Finally, the cells were separated and pelleted by centrifugation (20 min, 5000×g, 4°C) and discarded. After the supernatant was filtered through a 0.45 μm filter, 1/4 volume of 20% (w/v) PEG8000, 2.5M NaCl solution was added and added to Phage particles were pelleted by incubation in an ice bath for 1 hour. Then the pellet was centrifuged (20 min, 8000×g, 4°C), the supernatant was discarded, and the phage was resuspended in 25 ml of pre-cooled PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ ), Centrifuge (5 minutes, 20000 xg, 4°C). 1/4 volume of 20% (w/v) PEG8000, 2.5M NaCl solution was added to the supernatant and the phage particles were re-precipitated in an ice bath for 30 minutes. The pellet was centrifuged (30 min, 20000 xg, 4°C), the phage pellet was resuspended again in 2 ml of pre-chilled PBS, kept on ice for 30 min and centrifuged (30 min, 17000 xg, 4°C). The supernatant was mixed 1:1 with 4% (w/v) BSA in PBS, placed on a rotary mixer, incubated at room temperature for 30 minutes, and used directly for screening.

Example 3. Identification of 7B3 mutants that specifically bind to EGFRvIII

After four rounds of screening against the antigen EGFRvIII, 96 clones were randomly selected from the fourth round of screening and analyzed by single phage ELISA (enzyme-linked immunosorbent assay) against the antigens EGFRvIII and N1N2-806 (purchased from Ruijin Biotech) , wherein N1N2-806 is a fusion protein of M13 phage PIII protein N1N2 domain and amino acids 287-302 of EGFR. For this purpose, each single colony was inoculated with 300 μl of 2YT/ampicillin medium (containing 2% glucose) in a 96-well deep-well culture plate and cultured with shaking at 37° C. and 250 rpm for 16 hours. 500 μl of 2YT/ampicillin medium (containing 0.1% glucose) was inoculated with 20 μl of the culture, and cultured with shaking at 37° C. and 250 rpm for 1.5 hours. To prepare a helper phage solution, 75 μl of M13KO7 (titer of 3×10 ¹² pfu/ml) was mixed into 15 ml of 2YT medium, and 50 μl/well was added to the culture plate. Incubate at 37°C and 150rpm for 30 minutes, then add 50μl/well of the prepared kanamycin solution (take 180μl of 50mg/ml kanamycin, add to 15ml 2YT medium), shake at 37°C and 250rpm Incubate for 16 hours. Finally, the cells were pelleted by centrifugation (30 minutes, 5000 xg, 4°C), and the supernatant was transferred to a new 96-well deep-well culture plate.

For single phage ELISA, 100ng/well antigens EGFRvIII, N1N2-806 and negative control proteins BSA and N1N2 (purchased from Ruijin) were used on a 96-well MediSorp ELISA plate (purchased from Nunc), 50 μl/well, respectively, at 4 ℃ coated overnight. Each well was blocked with PBST containing 2% BSA (w/v). The wells were then washed three times with PBST and drained. Then 100 μl/well of each phage solution prepared above was added to each well of the plate. After incubation at 37°C for 2 hours, the cells were washed three times with PBST. To detect bound phage, anti-M13 antibody superoxide dismutase conjugate (purchased from GE Healthcare) was diluted 1:5000 in PBST and 100 μl was added to each well. The wells were rinsed three times with PBST and then three times with PBS after 1 hour incubation at 37°C. Finally, 50 μl of TMB substrate was pipetted into the wells, and the color was developed for 10 minutes at room temperature, followed by the addition of 50 μl of 2M H ₂ SO ₄ per well to stop the color reaction. Extinction values were measured at 450 nm with an enzyme-linked immunosorbent assay (Bio-Rad).

The clones with strong binding signal to antigen in ELISA but no binding signal to BSA were selected for subsequent evaluation and sequencing analysis. Subsequently, the antibody obtained from the light chain affinity maturation library and the antibody obtained from the heavy chain affinity maturation library in Example 2 were further combined in the light chain variable region and heavy chain variable region sequences, and the obtained antibody was also capable of specificity. The binding antigens EGFRvIII and N1N2-806 did not bind to the control proteins BSA and N1N2.

Considering the crystal structure of the antibody and antigenic determinants, the combination of the 7B3 light and heavy chain mutated antibody with EGFR _287-302 was structurally analyzed, and finally some amino acid sites were selected for further mutation to obtain higher affinity and stability . All altered amino acid sites include S31 in the light chain CDR1 region, V89, A92, Q93, F94 and Y96 in the light chain CDR3 region, S182 in the heavy chain CDR2 region, L222, R224, G225, F226 in the heavy chain CDR3 region and R227. On the basis of the combined antibody sequence after light and heavy chain mutation, the mutation site was further introduced to obtain antibody Y022. Compared with the parental antibody 7B3, Y022 contains 12 amino acid mutation sites (S31V, V89N, A92E, Q93N, F94I, Y96L, S182Q, L222M, R224K, G225N, F226W, R227D). As shown in Figure 1, in the single phage ELISA experiment, Y022 can specifically bind to the antigens EGFRvIII and N1N2-806, but not to the control proteins BSA and N1N2.

Nucleotide sequence of single chain antibody Y022 (717 bases; SEQ ID NO: 12):

Amino acid sequence of single chain antibody Y022 (239 amino acids; SEQ ID NO: 13):

Wherein positions 1-108 are light chain, and light chain CDR1 sequence: HASQDINVNIG (SEQ ID NO: 41), CDR2 sequence: HGKNLED (SEQ ID NO: 42), CDR3 sequence: NQYENIPLT (SEQ ID NO: 43).

Wherein positions 124-239 are heavy chain, and heavy chain CDR1 sequence: GYSITSDYAWN (SEQ ID NO: 44), CDR2 sequence: YISYRGRTQYNPSLKS (SEQ ID NO: 45), CDR3 sequence: MGKNWDY (SEQ ID NO: 46).

The nucleotide sequence of Y022 was contained in pCantab 5E due to screening from the previously constructed mutant library and site-directed mutagenesis, and the plasmid was named pCantab 5E-Y022 plasmid.

Using the same method used to generate antibody Y022, the inventors also obtained another 10 antibody clones with significantly improved affinity and stability, namely M14, M15, M25, M26, S7, S8, S17, S22, S23 and S29 . Compared with the parental antibody 7B3, the amino acid mutation sites contained in all single-chain antibodies are shown in Table 1.

Table 1

Antibody amino acid mutation site Y022 S31V, V89N, A92E, Q93N, F94I, Y96L, S182Q, L222M, R224K, G225N, F226W, R227D M14 V89N, A92E, Q93N, F94N, Y96I, S182N M15 S31V, V89N, A92E, Q93N, F94N, Y96I M25 S31V, V89N, A92E, Q93N, F94I, Y96L, S182R M26 S31V, V89N, A92E, Q93N, F94I, Y96L, S182Q S7 S31V, K53T, V89N, A92E, Q93N, F94N, Y96I S8 S31V, A44S, V89N, A92E, Q93N, F94N, Y96I S17 S31T, V89N, A92E, Q93N, F94N, Y96L, S182Q S22 S31V, K53T, V89N, A92E, Q93N, F94N, Y96L, S182R S23 S31V, A44S, V89N, A92E, Q93N, F94N, Y96L, S182R S29 S31V, V89N, A92E, Q93N, Y96L, S182R

Nucleotide sequence of single chain antibody M14 (717 bases; SEQ ID NO: 58):

Amino acid sequence of single chain antibody M14 (239 amino acids; SEQ ID NO: 59):

The amino acid sequences of M14 light chain CDR1 (HASQDINSNIG), CDR2 (HGKNLED), CDR3 (NQYENNPIT) and heavy chain CDR1 (GYSITSDYAWN), CDR2 (YISYRGRTNYNPSLKS), CDR3 (LGRGFRY) are SEQ ID NOs: 47, 42, 48, 44, respectively, 49, 50.

Nucleotide sequence of single chain antibody M15 (717 bases; SEQ ID NO: 60):

Amino acid sequence of single chain antibody M15 (239 amino acids; SEQ ID NO: 61):

The amino acid sequences of M15 light chain CDR1 (HASQDINVNIG), CDR2 (HGKNLED), CDR3 (NQYENNPIT) and heavy chain CDR1 (GYSITSDYAWN), CDR2 (YISYRGRTSYNPSLKS), CDR3 (LGRGFRY) are SEQ ID NOs: 41, 42, 48, 44, respectively, 51, 50.

Nucleotide sequence of single chain antibody M25 (717 bases; SEQ ID NO: 62):

Amino acid sequence of single chain antibody M25 (239 amino acids; SEQ ID NO: 63):

The amino acid sequences of M25 light chain CDR1 (HASQDINVNIG), CDR2 (HGKNLED), CDR3 (NQYENIPLT) and heavy chain CDR1 (GYSITSDYAWN), CDR2 (YISYRGRTRYNPSLKS), CDR3 (LGRGFRY) are SEQ ID NOs: 41, 42, 43, 44, respectively, 52, 50.

Nucleotide sequence of single chain antibody M26 (717 bases; SEQ ID NO: 64):

Amino acid sequence of single chain antibody M26 (239 amino acids; SEQ ID NO: 65):

The amino acid sequences of M26 light chain CDR1 (HASQDINVNIG), CDR2 (HGKNLED), CDR3 (NQYENIPLT) and heavy chain CDR1 (GYSITSDYAWN), CDR2 (YISYRGRTQYNPSLKS), CDR3 (LGRGFRY) are SEQ ID NOs: 41, 42, 43, 44, respectively, 45, 50.

Nucleotide sequence of single chain antibody S7 (717 bases; SEQ ID NO: 66):

Amino acid sequence of single chain antibody S7 (239 amino acids; SEQ ID NO: 67):

The amino acid sequences of S7 light chain CDR1 (HASQDINVNIG), CDR2 (HGTNLED), CDR3 (NQYENNPIT) and heavy chain CDR1 (GYSITSDYAWN), CDR2 (YISYRGRTSYNPSLKS), CDR3 (LGRGFRY) are SEQ ID NOs: 41, 53, 54, 44, respectively, 51, 50.

Nucleotide sequence of single chain antibody S8 (717 bases; SEQ ID NO: 68):

Amino acid sequence of single chain antibody S8 (239 amino acids; SEQ ID NO: 69):

The amino acid sequences of S8 light chain CDR1 (HASQDINVNIG), CDR2 (HGKNLED), CDR3 (NQYENNPIT) and heavy chain CDR1 (GYSITSDYAWN), CDR2 (YISYRGRTSYNPSLKS), CDR3 (LGRGFRY) are SEQ ID NOs: 41, 42, 54, 44, respectively, 51, 50.

Nucleotide sequence of single chain antibody S17 (717 bases; SEQ ID NO: 70):

Amino acid sequence of single chain antibody S17 (239 amino acids; SEQ ID NO: 71):

The amino acid sequences of S17 light chain CDR1 (HASQDINTNIG), CDR2 (HGKNLED), CDR3 (NQYENNPLT) and heavy chain CDR1 (GYSITSDYAWN), CDR2 (YISYRGRTQYNPSLKS), CDR3 (LGRGFRY) are SEQ ID NOs: 55, 42, 56, 44, respectively, 45, 50.

Nucleotide sequence of single chain antibody S22 (717 bases; SEQ ID NO: 72):

Amino acid sequence of single chain antibody S22 (239 amino acids; SEQ ID NO: 73):

The amino acid sequences of S22 light chain CDR1 (HASQDINVNIG), CDR2 (HGTNLED), CDR3 (NQYENNPLT) and heavy chain CDR1 (GYSITSDYAWN), CDR2 (YISYRGRTRYNPSLKS), CDR3 (LGRGFRY) are SEQ ID NOs: 41, 53, 56, 44, respectively, 52, 50.

Nucleotide sequence of single chain antibody S23 (717 bases; SEQ ID NO: 74):

Amino acid sequence of single chain antibody S23 (239 amino acids; SEQ ID NO: 75):

The amino acid sequences of S23 light chain CDR1 (HASQDINVNIG), CDR2 (HGKNLEDG), CDR3 (NQYENNPLT) and heavy chain CDR1 (GYSITSDYAWN), CDR2 (YISYRGRTRYNPSLKS), CDR3 (LGRGFRY) are SEQ ID NOs: 41, 42, 56, 44, respectively, 52, 50.

Nucleotide sequence of single chain antibody S29 (717 bases; SEQ ID NO: 76):

Amino acid sequence of single chain antibody S29 (239 amino acids; SEQ ID NO: 77):

The amino acid sequences of S23 light chain CDR1 (HASQDINVNIG), CDR2 (HGKNLED), CDR3 (NQYENFPLT) and heavy chain CDR1 (GYSITSDYAWN), CDR2 (YISYRGRTRYNPSLKS), CDR3 (LGRGFRY) are SEQ ID NOs: 41, 42, 57, 44, respectively, 52, 50.

Example 4. Expression and purification of antibodies

The gene of each antibody was inserted into the NdeI/XhoI site of the expression vector pET22B(+), and the antibody protein was recombinantly prepared in E. coliBL21 (DE3), and purified by nickel column with 6× histidine polypeptide fused at the carboxyl terminus. Specifically, to prepare antibody proteins, 5 ml of 2×YT/ampicillin medium was inoculated with each single colony, and cultured with shaking at 37° C. and 220 rpm for 16 hours. 100 ml of 2xYT/ampicillin medium was inoculated with 1 ml of this preculture and cultured with shaking at 37°C and 220 rpm until the cell density reached _OD600 = 0.5. After adding 1 mM α-D-isopropylthiogalactoside (IPTG) to induce foreign gene expression, the culture was continued at 30° C. and 220 rpm for 6 hours with shaking. Cells were then pelleted by centrifugation (15 min, 3500 xg, 4°C) and resuspended in 35 ml of disruption buffer (50 mM PB, 300 mM NaCl, 2 M urea, 0.5% Triton X-100, pH 8.0). After sonication, the samples were shaken at room temperature for 30 minutes to completely dissolve cell debris. The inclusion body pellet was then collected by centrifugation (15 min, 10,000 xg, 4°C), 20 ml of denaturing buffer (50 mM PB, 300 mM NaCl, 8 M urea, 10 mM imidazole, pH 8.0) was added and shaken at room temperature for one hour. The precipitate was removed by centrifugation (15 min, 10,000×g, 4° C.), the lysed supernatant was collected, and the protein was purified with a 5 ml HisTrap HP purification column (purchased from GE Healthcare). The purity of the purified antibody protein was analyzed by SDS polyacrylamide gel electrophoresis, and the protein concentration was determined by BCA method.

Example 5. Determination of binding activity of antibodies

The binding activity of the antibody to the antigen EGFRvIII was determined by a concentration gradient ELISA experiment. For this purpose, the antigen EGFRvIII was diluted with 0.1 M NaHCO ₃ (pH 9.6) coating solution, 200 ng per well, 50 μl/well, overnight at 4°C, and PBST containing 2% (w/v) BSA at room temperature closed for 2 hours. Plates were then rinsed three times with PBST and removed. Subsequently, 100 μl of each antibody protein in PBST at a range of concentrations (starting concentration 50 ng/well, 18 nM, until diluted to 1:81) in PBST was added to each well plate, and each sample was assayed using a triplicate well assay. After incubation at 37°C for 2 hours, the cells were rinsed three times with PBST, and then 100 μl/well of mouse anti-His-tag antibody (purchased from Santa Cruz) diluted 1:2000 was added and reacted at 37°C for 1 hour. To detect bound antibody, HRP-labeled goat anti-mouse antibody (purchased from Santa cruz) was diluted 1:15000 in PBST and 100 μl was added to each well and incubated at 37°C for 1 hour. For detection, wells were rinsed three times with PBST, then three times with PBS, and finally TMB was added to visualize for 15 min, the color reaction was stopped with 50 μl per well of 2M _H2SO4 , and the color reaction was stopped with an enzyme _- linked immunosorbent assay (Bio-Rad) at 450 nm. Measure the extinction value. The obtained absorbance intensity values were evaluated with Sigma Plot software, and the binding intensity of the antibody was calculated. For this purpose, the measured extinction value in each case was plotted against the corresponding antibody concentration and the resulting curve was fitted with the following nonlinear regression.

where the binding/dissociation equilibrium between the immobilized antigen and antibody protein is identified as:

x = concentration of antibody protein;

y=concentration of antigen/antibody complex (indirectly measured by absorbance after color reaction);

a = total concentration of immobilized antigen;

b = dissociation constant (K _D ).

The binding curve of antibody 7B3 obtained in the concentration gradient ELISA experiment is exemplarily shown in Figure 2, and its K _D value is about 22.4nM; the binding curve of antibody Y022 to EGFRvIII is shown in Figure 3, and its apparent K _D value is about 2.7 nM.

Example 6. Activity analysis of Y022 binding to cell surface EGFR

1. Expression and purification of scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc fusion antibodies

The scFv-Y022 fragment was amplified from the resulting clone using primer pairs V5-Y022-F (SEQ ID NO: 14, ACAGTGCTAGCAGATATTCAGATGACCCAG) and V5-Y022-R (SEQ ID NO: 15, AAGAATGCGGCCGCGCTGCTGCTCACGGTCACCAG) according to standard protocols; primers were used; scFv-806 was amplified from V5-806-F (SEQ ID NO: 16, ACAGTGCTAGCAGACATCCTGATGACCCAAT) and V5-806-R (SEQ ID NO: 17, AAGAATGCGGCCGCTGCAGAGACAGTGACCAG) using pH-806/CD3 (see 201210094008.X) as template Fragment; primer pairs V5-C225-F (SEQ ID NO: 18, ACAGTGCTAGCAGACATCTTGCTGACTCAG) and V5-C225-R (SEQ ID NO: 19, AAGAATGCGGCCGCTGCAGAGACAGTGACCAG) were used to create a C225 (VL-linker-VH) DNA fragment (sequence according to patent US20090099339A1) SEQ ID NO: 10 and SEQ ID NO: 12 were determined, obtained by Shanghai Ruijin Biotechnology Co., Ltd. through whole gene synthesis.) The scFv-C225 fragment was amplified as the template; the amplified product passed NheI/NotI (purchased from NEB) Double digestion, with T4 DNA ligase (purchased from NEB) in the same NheI/NotI double digestion vector plasmid pCMV-V5-Fc (the vector is fused downstream of the multi-cloning site to express the Fc fragment of human antibody IgG1, hereinafter referred to as V5 -Fc, purchased from Shanghai Ruijin Biotechnology Co., Ltd.) was connected and transformed into the host bacteria TOP10, and the clones were picked to identify positive clones by PCR and confirmed by sequencing to obtain V5-scFv-Y022-Fc, V5-scFv-806 - Fc and V5-scFv-C225-Fc eukaryotic expression plasmids.

The above expression plasmids were respectively transfected into well-grown HEK-293F cells, cultured at 37°C, 5% CO ₂ , 125 rpm shaker for 7 days, centrifuged at 4000 rpm for 10 min, removed the precipitate, collected the supernatant, and filtered with a 0.45 μm filter. The processed samples were subjected to affinity purification with protein A (purchased from GE) affinity column, and finally purified antibody-Fc fusion proteins scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc were obtained. The identification results are as follows shown in Figure 4.

2. FACS detection of the binding ability of single chain antibodies scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc to cell surface EGFR

The binding ability of each of the single chain antibodies scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc to the following cell lines was analyzed by fluorescence-activated cell sorter (FACS) (BD, FACS Calibur).

The specific method is as follows:

1) Take each tumor cell in the logarithmic growth phase as listed in Table 2 and inoculate it into a 6 cm dish, the inoculated cell density is about 90%, and culture it overnight in a 37°C incubator.

2) Digest the cells with 10 mM EDTA and collect the cells by centrifugation at 200 g × 5 min. Resuspend in 1% phosphate buffered saline (NBS PBS) containing calf serum at a concentration of 1×10 ⁶ to 1×10 ⁷ /mL, and add 100ul/tube into a dedicated flow-through tube.

3) Centrifuge at 200g × 5min and discard the supernatant.

4) Add the test antibodies scFv-Y022-Fc, scFv-806-Fc and scFv-C225-Fc respectively, while using PBS as a negative control, the final antibody concentration is 20 μg/ml, and 100 ul is added to each tube. Ice bath, 45 minutes.

5) Add 2ml of 1% NBS PBS to each tube, and centrifuge at 200g×5min for a total of two times.

6) Discard the supernatant, add FITC fluorescently labeled goat anti-human antibody (from Shanghai Kangcheng Bioengineering Co., Ltd.) diluted 1:50, and add 100 ul to each tube. Ice bath, 45 minutes.

7) Add 2 ml of 1% NBS PBS to each tube, and centrifuge at 200 g × 5 min for a total of two times.

8) Discard the supernatant, resuspend in 300ul of 1% NBS PBS, and detect by flow cytometry.

9) Analyze the data using the flow cytometry data analysis software WinMDI 2.9.

Table 2

The results are shown in Figure 5, the single-chain antibody of the present invention Y022 and the exogenous EGFR-overexpressing U87-EGFR (construction method according to: Wang H., et al., Identification of an Exon 4-Deletion Variant of Epidermal Growth Factor Receptor with Increased Metastasis-PromotingCapacity.Neoplasia, 2011, 13, 461-471) and U87-EGFRvIII overexpressing EGFRvIII (construction method according to: WO/2011/035465), endogenous overexpressing EGFR A431, CAL 27 and MDA-MB- 468 cells have different degrees of binding, especially with U87-EGFRvIII and A431 cells, but the binding ability is not as high as that of single chain antibody 806. The single-chain antibody C225 has a very strong binding ability to the above cells. These single chain antibodies had little binding to U87 cells.

In addition, both single-chain antibodies, Y022 and 806, were barely able to bind to the glioma cell line U87 cells. It is particularly noteworthy that single-chain antibody Y022 does not bind to normal prostate epithelial cells RWPE-1 and human primary keratinocyte K2, while single-chain antibody 806 binds to these two normal cells to varying degrees.

These results indicated that the single-chain antibody Y022 could specifically bind to tumor cells overexpressing EGFR and EGFRvIII, but hardly to normal cells expressing EGFR.

Example 7. Construction of an expression vector comprising a nucleotide sequence encoding a Y022/CD3 single-chain diabody

Taking the pCantab 5E-Y022 plasmid obtained in Example 3 as the template, using the forward primer pH7B3f2_fw (SEQ ID NO: 20, GATATTCAGATGACCCAGAGCCCGAGCAG) and the reverse primer pH7B3f2_re (SEQ ID NO: 21, AATAGGATCCACCACCTCCGCTGCTCACGGTCAC) as the primer pair, Y022scFv was obtained by PCR amplification DNA fragments. Another DNA fragment containing the signal peptide sequence of the pH carrier was obtained by PCR, using the pH-7B3/CD3 plasmid (see 201210094008.X Example 3 and Figure 2) as the template, and using the forward primer pH7B3f1_fw (SEQ ID NO: 22) , CCATTGACGCAAATGGGCGGTAGG) and the reverse primer pH7B3f1_re (SEQ ID NO: 23, CTGCTCGGGCTCTGGGTCATCTGAATATC). The two fragments were mixed in an equimolar ratio, fragment splicing and PCR were performed. The splicing conditions were: pre-denaturation: 94°C, 4min; denaturation: 94°C, 40s; annealing: 60°C, 40s; extension: 68°C, 140s, for 5 cycles, followed by a total extension at 68°C for 10 min. DNA polymerase and forward primer pH7B3f1_fw and reverse primer pH7B3f2_re were then supplemented, and amplified for 30 cycles. The amplification conditions were pre-denaturation: 94°C, 4min; denaturation: 94°C, 40s; 68°C, 140s, 30 cycles, then total extension 68°C, 10min.

The amplified sequences were simultaneously digested with restriction enzymes NheI/BamHI, and double digested according to the reaction conditions suggested by the enzyme supplier (NewEngland Biolabs, NEB). The expression vector pH (see 201210094008.X Example 3 and Figure 2) was similarly digested with restriction enzymes NheI/BamHI. Then, according to the reaction conditions suggested by the enzyme supplier (NEB), the double-digested Y022 scFv fragment and the pH vector fragment were ligated with T4 DNA ligase. The nucleotide sequence encoding the Y022 single-chain antibody polypeptide thus obtained is cloned into the vector, and together with the nucleotide sequence encoding the CD3 single-chain antibody polypeptide already contained in the vector, it can be transcribed into an mRNA, and finally translated into Y022/ CD3 single chain diabody polypeptide. The new plasmid was named pH-Y022/CD3, and its detailed structure is shown in Figure 6.

Example 8. Expression and purification of single-chain bifunctional antibodies Y022/CD3, pH-806/CD3 and pH-C225/CD3

The expression vectors pH-Y022/CD3, pH-806/CD3 and pH-C225/CD3 (see 201210094008.X) were respectively transfected into Chinese hamster ovary (CHO) cells according to the instructions of FreeStyle MAX Reagent (from Invitrogen) Stable clones were then screened according to the OptiCHO ^™ protein expression kit (from Invitrogen). Stable clones of CHO cells transfected with one of the above expression vectors, respectively, were cultured in shake flasks at 37° C., 130 rpm for 7 days, and the medium used was CD OptiCHO (from Gibco). The culture supernatant was obtained by centrifugation and then stored at -20°C.

Protein purification was performed using a histidine affinity chromatography column (His Trap HP column, purchased from GE Healthcare) according to the manufacturer's protocol. Specifically, the column was equilibrated with buffer A (20 mM sodium phosphate pH7.4, 0.4 M NaCl), and then the cell culture supernatant (500 mL supernatant) was added to the column (1 mL) after PBS dialysis at a flow rate of 1 mL. 3ml/min. The column was then washed with 5 volumes of buffer A and 10 volumes of buffer A containing 50 mM imidazole to remove contaminants. The bound protein of interest was eluted with the same buffer A supplemented with 250 mM imidazole. All purification steps were performed at 4°C.

The purified single-chain bifunctional antibodies were detected by reducing SDS-PAGE. As shown in Figure 7, the molecular weights of these antibody molecules were all about 60kD, which was consistent with the molecular weight of single-chain bifunctional antibodies calculated from the amino acid sequence.

Example 9. Antigen-binding specificity analysis of single-chain bifunctional antibodies such as Y022/CD3

The binding ability of the single-chain bifunctional antibody Y022/CD3 to EGFR was analyzed by fluorescence-activated cell sorter (FACS) (BD Company, FACSCalibur).

The specific method is as follows:

1. Take the tumor cells in the logarithmic growth phase as listed in Table 2 above and inoculate them into a 6 cm dish with a cell density of about 90%, and culture them overnight in a 37°C incubator.

2. Digest the cells with 10 mM EDTA and collect the cells by centrifugation at 200 g × 5 min. Resuspend in 1% phosphate buffered saline (NBS PBS) containing calf serum at a concentration of 1×10 6 ^to 1×10 ⁷ /mL, and add 100ul/tube into a dedicated flow-through tube.

3. Centrifuge at 200g × 5min and discard the supernatant.

4. Add the antibody to be tested Y022/CD3 separately, and use the irrelevant antibody NGR/CD3 as a negative control. The final concentration of the antibody is 5 μg/ml, and 100ul is added to each tube. Ice bath, 45 minutes.

5. Add 2ml of 1% NBS PBS to each tube, and centrifuge at 200g×5min for a total of two times.

6. Discard the supernatant, add 1:50 diluted mouse anti-histidine tag antibody (from Shanghai Ruixing Gene Technology Co., Ltd.), and add 100ul to each tube. Ice bath, 45 minutes.

7. Add 2ml of 1% NBS PBS to each tube, and centrifuge at 200g×5min for a total of two times.

8. Discard the supernatant, add FITC fluorescently labeled goat anti-mouse antibody (from Shanghai Kangcheng Bioengineering Co., Ltd.) diluted 1:50, and add 100ul to each tube. Ice bath, 45 minutes.

9. Add 2ml of 1% NBS PBS to each tube, and centrifuge at 200g×5min for a total of two times.

10. Discard the supernatant, resuspend in 300ul of 1% NBS PBS, and detect by flow cytometry.

11. Analyze the data using the flow cytometer data analysis software WinMDI 2.9.

The results are shown in FIG. 8 , the bifunctional antibody Y022/CD3 of the present invention can bind to U87-EGFR, U87-EGFRvIII and A431 cells, but hardly bind to U87 and human keratinocytes. These results suggest that Y022/CD3 can specifically bind to mutated human EGFR and EGFR-overexpressing tumor cells, but not to normal EGFR-expressing tissues.

In addition, as shown in the figure, Y022/CD3 can also bind to human peripheral blood mononuclear cells (PBMC) or Jurkat cells (human peripheral blood leukemia T cells, CD3 positive), suggesting that the bifunctional antibody of the present invention can specifically bind to CD3 antigen binding on the surface of T cells.

According to the methods mentioned in Examples 7 and 8, respectively construct expression plasmids (replace Y022 in Examples 7 and 8 with antibodies of other mutant forms) and express and purify M14/CD3, M15/CD3, M25/CD3, M26 /CD3, S7/CD3, S8/CD3, S17/CD3, S22/CD3, S23/CD3, S29/CD3. According to the method of this example, the binding ability of these antibodies to U87-EGFRvIII overexpressing EGFRvIII and endogenous EGFR overexpressing CAL27 cells was determined, respectively. The above antibodies were all able to bind to these two cells, and their mean fluorescence intensity (MFI) values are shown in Table 13.

Table 13

Antibody U87MG-EGFRvIII CAL 27 PBS 1 3.11 M14 36.52 28.39 M15 37.86 29.43 M25 36.52 24.14 M26 41.42 24.58 S7 42.17 27.88 S8 38.54 29.96 S17 31.62 25.03 S22 31.34 24.58 S23 32.2 29.69 S29 34.6 25.71

Example 10. Biological activity analysis of single-chain diabodies such as Y022/CD3-cytotoxicity to various tumor cells

Peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy human donors using Ficoll (from Biochrom) density gradient centrifugation following standard procedures. After centrifugation, cells were washed with phosphate buffered saline (PBS) at a concentration of 0.1 M and resuspended in RPMI 1640 complete medium (Gibco) to adjust the cell concentration to 5×10 ⁵ /mL. PBMCs were used as effector cells in cytotoxicity experiments. Different tumor cells serve as target cells. The target cell concentration was adjusted to 5 x ¹⁰⁴ /mL with RPMI 1640 complete medium. The same volumes of target cells and effector cells were mixed so that the ratio of effector cells:target cells (E:T) was 10:1.

The mixed cell suspension was added to a 96-well plate at a volume of 75 μL/well. Then 25 μL of ten-fold serial dilutions from 1000 ng/mL to 0.1 ng/mL of the following reagents were added to each well:

(1) Y022/CD3 single chain bifunctional antibody (BiTe);

(2) RPMI 1640 complete medium (background control);

(3) NGR/CD3 single-chain bifunctional antibody (negative control, NGR is a neovascularization targeting peptide, which has no cross-binding site with EGFR. It was prepared according to conventional methods).

After 40 hours of incubation in a 37°C, 5% _CO2 incubator, according to the manufacturer's instructions, use Non-Radioactive Cytotoxicity Assaykit (from Promega) detects the cytotoxicity of antibodies.

The nonradioactive cytotoxicity assay is a colorimetric-based assay that can replace the 51Cr release assay. The assay quantitatively measures lactate dehydrogenase (LDH). LDH is a stable cytoplasmic enzyme that is released upon cell lysis in much the same way that 51Cr is released in radioactive assays. The released LDH medium supernatant can be detected by a 30-minute coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) to red formazan. The amount of red product produced is proportional to the number of cells lysed.

The five EGFR-related tumor cells listed in Table 3 below were used to analyze T cell tumors mediated by the diabody Y022/CD3 of the present invention and the NGR/CD3 single-chain diabody unrelated to EGFR as a control. lethality.

The killing rate (ie, % cytotoxicity) of tumor cells is based on The non-radioactive cytotoxicity detection G1780 product instruction manual provides the following formula to calculate:

in:

"Experimental" refers to the LDH release value produced by the addition of antibody/effector cells/target cells to the experimental wells,

"Spontaneous effector cell" refers to the spontaneous release of LDH from effector cells,

"Spontaneous target cell" refers to the release of LDH that occurs when the cell is untreated by other factors,

"Target cell maximum" is the LDH release resulting from complete lysis of target cells after treatment with 0.8% Triton X-100,

"Target cell maximal-target cell spontaneous" represents the release of LDH resulting from complete lysis of cells after external treatment.

table 3

The results in Table 3 above show that tumor cells expressing mutated EGFR and/or overexpressing EGFR, such as U87-EGFRvIII, U87-EGFR and A431, are specifically killed by T cells directed by the bifunctional specific antibody Y022/CD3.

Specifically, in the above tumor cell group treated with Y022/CD3, the minimum specific cytotoxicity was 32.1%, and the maximum reached 66.2%. However, the cytotoxicity of Y022/CD3 to U87 cells expressing low levels of EGFR and human primary keratinocytes was very low, 3.4% and 4.5%, respectively, which was significantly lower than that of the above-mentioned tumor cells expressing mutant EGFR and/or EGFR overexpressing. Cytotoxicity.

More specifically, the % cytotoxicity results of Y022/CD3 and control antibody NGR/CD3 against each tumor at different concentrations are shown in Tables 4-8 below.

Table 4

table 5

Table 6

Table 7

Table 8

In addition, using the same method to express and purify the following BiTe: M14/CD3, M15/CD3, M25/CD3, M26/CD3, S7/CD3, S8/CD3, S17/CD3, S22/CD3, S23/CD3, S29/CD3 was subjected to in vitro toxicity test analysis, and the results are shown in Figure 9.

As can be seen from Figure 9, tumor cells expressing mutated EGFR and/or overexpressing EGFR, such as U87-EGFRvIII, U87-EGFR and CAL27, will be affected by bifunctional specific antibodies M14/CD3, M15/CD3, M25/CD3, M26/CD3, S7/CD3, S8/CD3, S17/CD3, S22/CD3, S23/CD3, S29/CD3 antibody-directed T cells killed to varying degrees. However, U87 had almost no killing effect on cells expressing low levels of EGFR.

Example 11. Construction and viral packaging of a lentiviral plasmid expressing the chimeric antigen receptor protein encoded by the nucleic acid of the present invention

To construct the chimeric antigen receptor, the connection sequence of each part of the chimeric antigen receptor exemplified by the present invention is shown in Table 9 and FIG. 10 .

Table 9

Chimeric Antigen Receptor Extracellular binding domain - transmembrane domain - intracellular signal domain 1 - intracellular signal domain 2, etc. describe Y022-δZ scFv(EGFR)-CD8-CD3δzeta negative control Y022-Z scFv(EGFR)-CD8-CD3zeta first generation Y022-BBZ scFv(EGFR)-CD8-CD137-CD3zeta second generation Y022-28Z scFv(EGFR)-CD28a-CD28b-CD3zeta second generation Y022-28BBZ scFv(EGFR)-CD28a-CD28b-CD137-CD3zeta Third Generation

Note: CD28a represents the transmembrane region of CD28 molecule, and CD28b represents the intracellular signal region of CD28 molecule.

1. Amplification of nucleic acid fragments

(1) Amplification of scFv sequences

Using the pCantab 5E-Y022 plasmid as the template, the forward primer (SEQ ID NO: 24, including part of the sequence of CD8signal peptide) and the reverse primer (SEQ ID NO: 25, including part of the sequence of CD8hinge) were used as primer pairs, and PCR amplification was performed. Y022scFv was obtained by increasing it.

SEQ ID NO: 24

TGCTCCACGCCGCCAGGCCGGATATTCAGATGACCCAG

SEQ ID NO: 25

CGCGGCGCTGGCGTCGTGGTGCTGCTCACGGTCAC

(2) Nucleic acid sequences of other parts of the chimeric antigen receptor

The nucleic acid sequence of the anti-EGFRvIII chimeric antigen receptor protein except for Y022 scFv was obtained by PCR using the sequence SEQ ID NO: 26, 27, 28, 29 and 30 disclosed in the patent application number 201310164725.X respectively as templates . Specifically, the eGFP-F2A-CD8sp sequence was obtained by PCR amplification with the primer pair (SEQ ID NO: 26, 27) using the SEQ ID NO: 27 plasmid contained in Patent Application No. 201310164725.X as a template. CD8-CD3δzeta (δZ) was obtained by using the plasmid of SEQ ID NO: 26 in the patent application 201310164725.X as a template, and a primer pair (SEQ ID NO: 28, 29) was used to obtain it by PCR amplification. The sequences of CD8-CD3zeta(Z), CD8-CD137-CD3zeta(BBZ), CD28a-CD28b-CD3zeta(28Z) and CD28a-CD28b-CD137-CD3zeta(28BBZ) are respectively SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27, SEQ ID NO: 27) The plasmids corresponding to ID NO: 28, SEQ ID NO: 29 and SEQ ID NO: 30 were used as templates, and were obtained by PCR amplification using a primer pair (SEQ ID NO: 28, 30).

SEQ ID NO: 26

TGCAGTAGTCGCCGTGAAC

SEQ ID NO: 27

CGGCCTGGCGCGGCGTGGAGCA

SEQ ID NO: 28

ACCACGACGCCAGCGCCGCGACCAC

SEQ ID NO: 29

GAGGTCGACCTACGCGGGGGCGTCTGCGCTCCTGCTGAACTTCACTCT

SEQ ID NO: 30

GAGGTCGACCTAGCGAGGGGGGCAGGGCCTGCATGTGAAG

2. Splicing of nucleic acid fragments

The eGFP-F2A-CD8sp nucleic acid fragment obtained as described above was mixed with equimolar Y022scFv nucleic acid fragment and equimolar CD8-CD3δzeta (δZ) or CD8-CD3zeta (Z) or CD8-CD137-CD3zeta (BBZ) or CD28a- The CD28b-CD3zeta(28Z) or CD28a-CD28b-CD137-CD3zeta(28BBZ) nucleic acid fragment was spliced into three fragments and PCR was performed as shown in Figure 9. The splicing conditions were: pre-denaturation: 94°C, 4min; denaturation: 94°C, 40s ; Annealing: 60°C, 40s; Extension: 68°C, 140s, 5 cycles, then total extension at 68°C, 10min, supplemented with DNA polymerase and forward primer (SEQ ID NO: 24) and reverse primer (CD8- The reverse primer corresponding to CD3δzeta is SEQ ID NO: 29, and the rest are SEQ ID NO: 30), followed by 30 cycles of PCR amplification, and the amplification conditions were pre-denaturation: 94°C, 4min; : 60°C, 40s; extension: 68°C, 140s, 30 cycles, and then a total extension of 68°C, 10min. The fragments obtained by amplification are called (Table 2):

3. Construction of lentiviral plasmid vector

As an example, the vector system used in the lentiviral plasmid vector constructed below belongs to the third-generation self-inactivating lentiviral vector system. The system has three plasmids, namely, the encoding protein Gag/Pol and the packaging plasmid psPAX2 encoding the Rev protein (purchased from addgene). ); envelope plasmid PMD2.G (purchased from addgene) encoding VSV-G protein and recombinant expression vector encoding target gene CAR based on empty vector pWPT-eGFP (purchased from addgene).

In the empty vector pWPT-eGFP, the self-contained elongation factor-1α (EF-1α) promoter can regulate the expression of enhanced green fluorescent protein (eGFP). After inserting the construct previously constructed in this example, a recombinant expression vector encoding the target gene CAR is formed, wherein eGFP and the purpose are realized by the ribosomal skipping sequence (food and mouth disease virus, FMDV, ribosomal skipping sequence, F2A) from foot-and-mouth disease virus. Co-expression of the gene CAR. F2A is a core sequence of 2A (or "self-cleaving polypeptide 2A") derived from foot-and-mouth disease virus. It has the "self-cleaving" function of 2A and can achieve co-expression of upstream and downstream genes. 2A provides an effective and feasible strategy for constructing gene therapy polycistronic vectors due to its high shearing efficiency, high balance of upstream and downstream gene expression and short sequence. Especially in immunotherapy based on chimeric antigen receptor gene-modified T lymphocytes, this sequence is often used to achieve the co-expression of the target gene and GFP or eGFP, and the expression of CAR can be indirectly detected by detecting GFP or eGFP.

In this example, a lentiviral expression vector co-expressed by F2A-linked eGFP and a specific CAR was constructed, collectively referred to as pWPT-eGFP-F2A-CAR. The target gene eGFP-F2A-CAR obtained in the above-mentioned step 2 (see 2 in Example 7, the component behind F2A is referred to as CAR for short) is double-enzyme digested by MluI and SalI restriction endonucleases, and is connected into the same double-enzyme-cut. pWPT vector to construct a lentiviral vector expressing each chimeric antigen receptor. After the successfully constructed vector has been identified by MluI and SalI digestion and sequenced correctly, it can be ready for lentiviral packaging. As mentioned before, eGFP-F2A-CAR is transcribed into a single mRNA, but finally translated into two peptide chains of eGFP and anti-EGFRvIII chimeric antigen receptor, in which the anti-EGFRvIII chimeric antigen receptor will be localized under the guidance of the CD8α signal peptide on the cell membrane.

The obtained vector containing each purpose CAR is as follows (the component behind F2A can be referred to as CAR for short):

pWPT-eGFP-F2A-Y022scFv-SZ;

pWPT-eGFP-F2A-Y022scFv-Z;

pWPT-eGFP-F2A-Y022scFv-BBZ;

pWPT-eGFP-F2A-Y022scFv-28Z;

pWPT-eGFP-F2A-Y022scFv-28BBZ.

Through the above construction, five eGFP-F2A-CAR polypeptide sequences can be obtained respectively, which are called:

eGFP-F2A-Y022scFv-δZ (SEQ ID NO: 36);

eGFP-F2A-Y022scFv-Z (SEQ ID NO: 37);

eGFP-F2A-Y022scFv-BBZ (SEQ ID NO: 38);

eGFP-F2A-Y022scFv-28Z (SEQ ID NO: 39);

eGFP-F2A-Y022scFv-28BBZ (SEQ ID NO: 40).

4. Plasmid transfection of 293T packaging lentivirus

HEK-293T cells (ATCC: CRL-11268) cultured to passages 6 to 10 were seeded at a density of 6×10 ⁶ in 10 cm dishes at 37° C., 5% CO ₂ overnight to prepare for transfection. The medium is DMEM (purchased from PAA company) containing 10% fetal bovine serum (purchased from PAA company)

The steps of transfection are as follows:

4.1 Preparation of Solution A: Dissolve 10 μg mock control or 10 μg of each target gene plasmid pWPT-eGFP-F2A-CAR, 7.5 μg packaging plasmid PAX2: and 3 μg envelope plasmid pMD2.G, respectively, into 800 μL of serum-free DMEM medium medium, mix well.

4.2 Preparation of solution B: Dissolve 60 μg PEI (polyethyleneimine, purchased from Polysciences) in 800 μL of serum-free DMEM medium, mix gently, and incubate at room temperature for 5 min.

4.3 Formation of transfection complex: Add solution A to solution B and mix gently. Immediately after adding, vortex to mix or mix gently, and incubate at room temperature for 20 min.

4.4 1.6 ml of the transfection complex was added dropwise to HEK-293T cells. After 4-5 h, the medium of the transfected 293T cells was changed with 2% FBS in DMEM medium.

The transfection efficiency (ie, the proportion of cells with green fluorescence) was observed on the next day of transfection, and a positive transfection efficiency of ∼80% was considered a successful transfection experiment. After 72 hours of transfection, the virus was collected by filtration using a 0.45 μm filter (purchased from Millipore), and then centrifuged at 28,000 rpm using a Beckman Optima L-100XP ultracentrifuge at 4°C for 2 hours. AIM-V medium (purchased from Invitrogen) was resuspended with ~1/50 volume of the stock solution, aliquoted in 100 μL/tube and stored at -80°C until virus titration or infection of T lymphocytes.

5. Determination of lentivirus titers packaged with mock or eGFP-F2A-CAR

On the first day, 293T cells were seeded at 1×10 ⁵ /mL in a 96-well culture plate, 100 μL/well, at 37° C., 5% CO ₂ , and the culture medium was DMEM containing 10% fetal bovine serum. The next day, discard 50 μL/well of the culture supernatant, and add 50 μL/well of fresh culture medium containing polybrene with a final concentration of 6 μg/mL, incubate at 37°C, 5% CO ₂ for 30 min. Add 10 μL/well of virus stock solution or 1 μL/well of virus concentrate, 3-fold dilution, 6 gradients, two duplicate wells, 37°C, 5% CO ₂ for incubation. After 48 hours of infection, eGFP was detected by flow cytometry, and the number of cells with a positive rate of 5-20% was appropriate, and the calculated titer (U/mL)=positive rate×dilution factor×100×10 ⁴ . The titers of the above-mentioned viruses containing mock or empty vector control and each eGFP-F2A-CAR packaged by PEI transfection method are about 0.5-1×10 ⁷ U/mL, and the virus titers measured after concentration are about It is 0.5～1×10 ⁸ U/mL.

Example 12. Infection of T cells with recombinant lentivirus

Human peripheral blood mononuclear cells (provided by Shanghai Blood Center) were obtained from the peripheral blood of healthy people by density gradient centrifugation, and cultured by adding AIM-V lymphocyte medium (purchased from Invitrogen Company) at a density of about 2×10 ⁶ /mL, Magnetic beads (Invitrogen) coated with anti-CD3 and CD28 antibodies were added at a cell:magnetic bead ratio of 1:1, and recombinant human IL-2 (purchased from Shanghai Huaxin Biotech Co., Ltd.) was added at a final concentration of 300 U/mL. Company) were stimulated and cultured for 48 h, and then T cells were infected with the above-mentioned recombinant lentivirus (MOI≈15). The expression of different chimeric antigen receptors was detected by flow cytometry in the infected T cells on the 8th day of culture. Since eGFP and CAR were co-expressed, the positive cells that detected eGFP were positive cells that expressed chimeric antigen receptors. Using uninfected T lymphocytes as a negative control, the positive rates of virus-infected T cells expressing different chimeric antigen receptors are shown in Table 10. The results of the positive rate indicated that a certain positive rate of CART cells could be obtained by lentivirus infection.

Table 10

T cells transfected with the following CARs CAR T cell eGFP positive rate Y022-δZ(Mock) 66% Y022-Z 58% Y022-BBZ 53% Y022-28Z 54% Y022-28BBZ 52%

After T cells were infected with viruses packaged with different chimeric antigen receptors, they were subcultured at a cell density of 5×10 ⁵ /ml every other day, counted, and supplemented with IL-2 (final concentration of IL-2). 300U/ml), about 100-1000-fold expansion on the 11th day of culture, indicating that T cells expressing different chimeric antigen receptors can be expanded in vitro to a certain amount, which provides a basis for subsequent in vitro toxicity tests and in vivo tests. ensure.

Example 13. In vitro antitumor activity of CAR-Y022

The materials used in the in vitro toxicity experiments are as follows:

The target cells were U87, U87-EGFR, U87-EGFRvIII, A431, CAL 27, MDA-MB-468, RWPE-1 cells and human primary keratinocyte K2 as shown in Table 5, respectively. The effector cells were T lymphocytes (CAR T cells) positive for chimeric antigen receptor expression detected by FACS cultured for 12 days in vitro.

The effector-target ratios were 3:1, 1:1 and 1:3 respectively, the number of target cells was 10000/well, and each group had 5 duplicate wells. The detection time is 18h.

The experimental groups and the control groups are as follows:

Each experimental group: each target cell + CAR T lymphocytes expressing different chimeric antigen receptors;

Control group 1: the target cells release the maximum LDH;

Control group 2: target cells spontaneously release LDH;

Control 3: Effector cells spontaneously release LDH.

Detection method: CytoTox 96 non-radioactive cytotoxicity detection kit (Promega company) was used. This method is a colorimetric-based detection method that can replace the 51Cr release method. The assay quantitatively measures lactate dehydrogenase (LDH). LDH is a stable cytoplasmic enzyme that is released upon cell lysis in much the same way that ⁵¹ Cr is released in radioactive assays. The released LDH medium supernatant can be detected by a 30-minute coupled enzymatic reaction in which LDH converts a tetrazolium salt (INT) to red formazan. The amount of red product produced is proportional to the number of cells lysed. For details, please refer to the instructions of CytoTox96 non-radioactive cytotoxicity detection kit.

The formula for calculating cytotoxicity is:

Specifically as shown in Table 11 and Table 12, in the case of different effector-target ratios, compared with 806-CAR T, the Y022-28Z CAR T and Y022-28BBZ CAR T expressing the chimeric antigen receptor of the present invention are more efficient than 806-CAR T. Cells expressing EGFR and EGFRvIII have obvious killing effect, and show a gradient-dependent effect of target ratio. The higher the immediate effect target ratio, the stronger the cytotoxic effect. The effector-target ratio-dependent data further showed the specific cytotoxic effect of CART cells expressing the chimeric antigen receptor of the present invention on cells that highly express EGFR and its variants.

It is worth noting that Y022-CAR T has almost no killing effect on RWPE-1 cells that normally express EGFR and human primary keratinocyte K2. When the effector-target ratio is 3:1, the chimeric antigen receptor Y022-28BBZ CAR T lymphocytes The cytotoxicity of the cells to RWPE-1 cells and human primary keratinocyte K2 was 12% and 2%, respectively, and the cytotoxicity of Y022-28Z CAR T lymphocytes to RWPE-1 cells and human primary keratinocyte K2 was 8%, respectively and 3%. In contrast, 806-CAR T had different degrees of killing on these two cells, and the cytotoxicity of 806-28BBZ CAR T lymphocytes against RWPE-1 cells and human primary keratinocyte K2 was 25% and 22%, respectively. , 806-28Z CAR T lymphocytes were 15% and 13% cytotoxic to RWPE-1 cells and human primary keratinocyte K2, respectively.

In addition, CART as a negative control transfected with a virus containing a mock plasmid (carrying scFv-Y022-δZ) showed very low cytotoxicity to all of the above cell lines.

The above results show that the chimeric antigen receptor Y022-CART constructed by the single-chain antibody against EGFR and its variants can selectively kill tumor cells with high expression of EGFR and its variants (EGFRvIII), while the normal expression of EGFR cells have almost no killing effect. In addition, from the cytotoxicity data, the third-generation (Y022-28BBZ) CAR T was more toxic to target cells than the second-generation (Y022-28Z) CAR T.

Table 11

Table 12

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (43)

1. an antibody that specifically recognizes the EGFRvШ expressed by tumor cells or the EGFR overexpressed, is characterized in that, comprises: Antibody (a), its light chain variable region has CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:42, and CDR3 shown in SEQ ID NO:43; its heavy chain variable region has SEQ ID CDR1 shown in NO:44, CDR2 shown in SEQ ID NO:45, CDR3 shown in SEQ ID NO:46; Antibody (b), its light chain variable region has CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:42, CDR3 shown in SEQ ID NO:48; its heavy chain variable region has SEQ ID CDR1 shown in NO:44, CDR2 shown in SEQ ID NO:49, CDR3 shown in SEQ ID NO:50; Antibody (c), its light chain variable region has CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:42, and CDR3 shown in SEQ ID NO:48; its heavy chain variable region has SEQ ID CDR1 shown in NO:44, CDR2 shown in SEQ ID NO:51, CDR3 shown in SEQ ID NO:50; Antibody (d), its light chain variable region has CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:42, and CDR3 shown in SEQ ID NO:43; its heavy chain variable region has SEQ ID CDR1 shown in NO:44, CDR2 shown in SEQ ID NO:52, CDR3 shown in SEQ ID NO:50; Antibody (e), its light chain variable region has CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:42, CDR3 shown in SEQ ID NO:43; its heavy chain variable region has SEQ ID CDR1 shown in NO:44, CDR2 shown in SEQ ID NO:45, CDR3 shown in SEQ ID NO:50; Antibody (f), its light chain variable region has CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:53, CDR3 shown in SEQ ID NO:54; its heavy chain variable region has SEQ ID CDR1 shown in NO:44, CDR2 shown in SEQ ID NO:51, CDR3 shown in SEQ ID NO:50; Antibody (g), its light chain variable region has CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:42, CDR3 shown in SEQ ID NO:54; its heavy chain variable region has SEQ ID CDR1 shown in NO:44, CDR2 shown in SEQ ID NO:51, CDR3 shown in SEQ ID NO:50; Antibody (h), its light chain variable region has CDR1 shown in SEQ ID NO:55, CDR2 shown in SEQ ID NO:42, and CDR3 shown in SEQ ID NO:56; its heavy chain variable region has SEQ ID CDR1 shown in NO:44, CDR2 shown in SEQ ID NO:45, CDR3 shown in SEQ ID NO:50; Antibody (i), its light chain variable region has CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:53, and CDR3 shown in SEQ ID NO:56; its heavy chain variable region has SEQ ID CDR1 shown in NO:44, CDR2 shown in SEQ ID NO:52, CDR3 shown in SEQ ID NO:50; Antibody (j), its light chain variable region has CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:42, CDR3 shown in SEQ ID NO:56; its heavy chain variable region has SEQ ID CDR1 shown in NO:44, CDR2 shown in SEQ ID NO:52, CDR3 shown in SEQ ID NO:50; or Antibody (k), its light chain variable region has CDR1 shown in SEQ ID NO:41, CDR2 shown in SEQ ID NO:42, CDR3 shown in SEQ ID NO:57; its heavy chain variable region has SEQ ID CDR1 shown in NO:44, CDR2 shown in SEQ ID NO:52, CDR3 shown in SEQ ID NO:50. 2. The antibody of claim 1, wherein the amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 13; the light chain of the antibody The amino acid sequence of the variable region is shown in positions 1-108 in SEQ ID NO: 13; The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO:59; the amino acid sequence of the variable region of the light chain of the antibody is shown in the first 1 in SEQ ID NO:59 -108 bits shown; The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO:61; the amino acid sequence of the variable region of the light chain of the antibody is shown in the first 1 in SEQ ID NO:61 -108 bits shown; The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO:63; the amino acid sequence of the variable region of the light chain of the antibody is shown in the first 1 in SEQ ID NO:63 -108 bits shown; The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO:65; the amino acid sequence of the variable region of the light chain of the antibody is shown in the first 1 in SEQ ID NO:65 -108 bits shown; The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO:67; the amino acid sequence of the variable region of the light chain of the antibody is shown in the first 1 in SEQ ID NO:67 -108 bits shown; The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO:69; the amino acid sequence of the variable region of the light chain of the antibody is shown in the first 1 in SEQ ID NO:69 -108 bits shown; The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO:71; the amino acid sequence of the variable region of the light chain of the antibody is shown in the first 1 in SEQ ID NO:71 -108 bits shown; The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 73; the amino acid sequence of the variable region of the light chain of the antibody is shown in the first 1 in SEQ ID NO: 73 -108 bits shown; The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO:75; the amino acid sequence of the variable region of the light chain of the antibody is shown in the first 1 in SEQ ID NO:75 -108 bits indicated; or The amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO:77; the amino acid sequence of the variable region of the light chain of the antibody is shown in the first 1 in SEQ ID NO:77 -108 bits shown. 3. The antibody of claim 1, wherein the antibody is antibody (a). 4. The antibody of claim 3, wherein the amino acid sequence of the variable region of the heavy chain of the antibody is shown in positions 124-239 in SEQ ID NO: 13; the light chain of the antibody The amino acid sequence of the variable region is shown in positions 1-108 of SEQ ID NO:13. 5. A nucleic acid encoding the antibody of any one of claims 1-4. 6. An expression vector comprising the nucleic acid of claim 5. 7. A host cell comprising the expression vector of claim 6 or the nucleic acid of claim 5 integrated into the genome. 8. the purposes of the arbitrary described antibody of claim 1-4, is used for the preparation of the targeted drug that specifically targets the tumor cell that expresses EGFRvШ or overexpressed EGFR, antibody drug conjugate or multifunctional antibody; Or For the preparation of reagents for diagnosing tumors expressing EGFRvШ or overexpressing EGFR; or For the preparation of chimeric antigen receptor-modified immune cells. 9. The use of claim 8, wherein the immune cells comprise: T lymphocytes or NK cells. 10. A multifunctional immunoconjugate, characterized in that, the multifunctional immunoconjugate comprises: The antibody of any one of claims 1-4; and A functional molecule connected to it; the functional molecule is selected from: a molecule targeting tumor surface markers, a tumor-inhibiting molecule, a molecule targeting immune cell surface markers or a detectable marker. 11. The multifunctional immunoconjugate of claim 10, wherein the molecule targeting a tumor surface marker is an antibody or a ligand that binds to the tumor surface marker; or The tumor-inhibiting molecules are anti-tumor cytokines or anti-tumor toxins. 12. The multifunctional immunoconjugate of claim 11, wherein the cytokines comprise: IL-12, IL-15, IFN-beta, and TNF-alpha. 13. The multifunctional immunoconjugate of claim 10, wherein the detectable label comprises: a chromogenic label. 14. The multifunctional immunoconjugate of claim 13, wherein the chromogenic label comprises: a fluorescent label. 15. The multifunctional immunoconjugate of claim 10, wherein the molecule targeting the surface marker of an immune cell is an antibody or a ligand that binds to the surface marker of an immune cell. 16. The multifunctional immunoconjugate of claim 15, wherein the immune cell surface markers comprise: CD3, CD16, and CD28. 17. The multifunctional immunoconjugate of claim 15, wherein the molecule targeting the surface marker of immune cells is an antibody that binds to the surface marker of T cells, which is consistent with claims 1-4. Any of the antibodies described form a T cell-engaged bifunctional antibody. 18. The multifunctional immunoconjugate of claim 17, wherein the antibody that binds to an immune cell surface marker is an anti-CD3 antibody. 19. The multifunctional immunoconjugate according to claim 10, wherein it is a fusion polypeptide, and between the antibody according to any one of claims 1-4 and the functional molecule linked to it, it also includes linking peptides. 20. A nucleic acid encoding the multifunctional immunoconjugate of any of claims 10-19. 21. Use of the multifunctional immunoconjugate of any one of claims 10-19, for the preparation of an antitumor drug, or For the preparation of reagents for diagnosing tumors expressing EGFRvШ or overexpressing EGFR; or For the preparation of chimeric antigen receptor-modified immune cells. 22. The use of claim 21, wherein the immune cells comprise: T lymphocytes or NK cells. 23. The chimeric antigen receptor comprising the antibody of any one of claims 1-4, which is expressed on the surface of an immune cell, wherein the chimeric antigen receptor comprises sequentially linked: claims 1-4 Any one of the antibodies, transmembrane region and intracellular signal region; the intracellular signal region is selected from: CD3ζ, FcεRIγ, CD27, CD28, CD137, CD134 intracellular signal region sequence, or a combination thereof. 24. The chimeric antigen receptor of claim 23, wherein the transmembrane region comprises the transmembrane region of CD8 or CD28. 25. The chimeric antigen receptor of claim 23, wherein the immune cells comprise: T lymphocytes or NK cells. 26. The chimeric antigen receptor of claim 23, wherein the chimeric antigen receptor comprises the following sequence-linked antibodies, a transmembrane region and an intracellular signal region: The antibody, CD8 and CD3ζ of any one of claims 1-4; The antibody, CD8, CD137 and CD3ζ of any one of claims 1-4; The antibody of any one of claims 1-4, the transmembrane region of the CD28 molecule, the intracellular signal region of the CD28 molecule, and CD3ζ; or The antibody of any one of claims 1-4, the transmembrane region of CD28 molecule, the intracellular signal region of CD28 molecule, CD137 and CD3ζ. 27. The chimeric antigen receptor of claim 23, wherein the antibody is a single chain antibody or a domain antibody. 28. The chimeric antigen receptor of claim 23, wherein the chimeric antigen receptor has: SEQ ID NO: 36 or the amino acid sequence shown at positions 285-601 therein; or SEQ ID NO: 37 or the amino acid sequence shown at positions 285-702 thereof; or SEQ ID NO: 38 or the amino acid sequence shown at positions 285-744 therein; or SEQ ID NO: 39 or the amino acid sequence shown at positions 285-749 therein; or SEQ ID NO: 40 or the amino acid sequence shown at positions 285-791 therein. 29. A nucleic acid encoding the chimeric antigen receptor of any of claims 23-28. 30. An expression vector comprising the nucleic acid of claim 29. 31. A virus, characterized in that the virus comprises the vector of claim 30. 32. Use of the chimeric antigen receptor of any one of claims 23-28, or the nucleic acid of claim 29, or the expression vector of claim 30, or the virus of claim 31, for use in Gene-modified immune cells targeting tumor cells expressing EGFRvШ or overexpressing EGFR were prepared. 33. A genetically modified immune cell, characterized in that it is transduced with the nucleic acid of claim 29, or the expression vector of claim 30 or the virus of claim 31; or The chimeric antigen receptor according to any one of claims 23-28 is expressed on its surface. 34. The immune cell of claim 33, further carrying a coding sequence for an exogenous cytokine. 35. The immune cell of claim 34, wherein the cytokine comprises: IL-12, IL-15 or IL-21. 36. The immunocyte of claim 33, wherein it also expresses another chimeric antigen receptor, the receptor does not contain CD3ζ, but contains the intracellular signaling domain of CD28, the intracellular signaling of CD137 domain or a combination of the two. 37. The immune cell of claim 33, further expressing a chemokine receptor. 38. The immune cell of claim 37, wherein the chemokine receptor comprises: CCR2. 39. The immune cell of claim 33, further expressing siRNA that can reduce PD-1 expression or a protein that blocks PD-L1. 40. The immune cell of claim 33, further expressing a safety switch. 41. The immune cell of claim 40, wherein the safety switch comprises: iCaspase-9, truncated EGFR or RQR8. 42. Use of the genetically modified immune cells according to any one of claims 33-41, characterized in that, for preparing a drug for inhibiting tumors, the tumor is a tumor expressing EGFRvШ or overexpressing EGFR. 43. A pharmaceutical composition, characterized in that it comprises: The antibody of any one of claims 1-4 or a nucleic acid encoding the antibody; or The immunoconjugate of any one of claims 10-19 or a nucleic acid encoding the conjugate; or The chimeric antigen receptor of any one of claims 23-28 or the nucleic acid encoding the chimeric antigen receptor; or The genetically modified immune cell of any one of claims 33-40.